Peptides useful for altering bone resorption

ABSTRACT

The present invention provides Arg--Gly--Asp peptides that can alter the binding of osteoclasts to a matrix such as bone or can selectively alter integrin receptor binding. The invention also provides methods of using the Arg--Gly--Asp peptides to alter α V  β 3  integrin receptor-mediated binding of a cell such as an osteoclast, endothelial cell or smooth muscle cell to a matrix. The invention further provides methods for ameliorating the severity of a pathology characterized, in part, by an undesirable level of bone resorption, angiogenesis or restenosis in a subject.

BACKGROUND OF THE INVENTION

This application is a continuation-in-part of U.S. Ser. No. 08/303,052,filed Sep. 8, 1994, which is a continuation-in-part of U.S. Ser. No.08/227,316, filed Apr. 13, 1994, now abandoned.

FIELD OF THE INVENTION

This invention relates generally to the field of medicine and morespecifically to peptides that can reduce or inhibit bone resorption,angiogenesis and restenosis.

Background Information

Bone is a dynamic tissue that is continually remodeled throughout lifedepending on factors such as nutrition and the load the bone must carry.Normal bone formation depends on the delicate balance between new boneaddition and old bone resorption. Bone resorption is initiated when anosteoclast attaches to the surface of mineralized bone, forms a tightsealing zone and secretes enzymes that begin the resorption process.After a period of several hours to days, the osteoclast detaches fromthe bone, leaving a pit on the bone surface. Under normal conditions,the pit is a target for osteoblasts, which deposit a material thatultimately becomes new bone.

Bone loss can result when the bone resorptive process is dominant overthe bone formative process. Such a condition can occur, for example,when a previously active person becomes sedentary or incapacitated dueto an injury or illness, when a person fails to ingest an adequateamount of vitamins and minerals or in various pathological conditions. Acondition of excessive bone loss, which is called osteoporosis, iscommon in elderly persons.

When osteoporosis is caused by a mineral or vitamin deficiency, thecondition often can be treated by supplementing the person's diet withthe appropriate nutrient. However, diet supplementation is not alwayseffective and, in most cases, osteoporosis cannot be treated. In severecases, osteoporosis can result in the formation of fragile bones thatreadily fracture when placed under a minimal load or stress. Thus, aneed exists for agents that can reduce or inhibit bone resorption in asubject or to achieve net bone accumulation. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides non-naturally occurring RGD-containingpeptides useful for reducing or inhibiting bone resorption, angiogenesisor restenosis and for altering an integrin receptor-mediatedinteraction. In addition, the invention provides pharmaceuticalcompositions comprising a non-naturally occurring RGD-containingpeptide.

The invention also provides methods for specifically altering α_(V) β₃integrin receptor-mediated binding of a cell to a matrix. The inventionprovides, for example, methods of reducing or inhibiting osteoclastbinding to a matrix, altering angiogenesis and reducing or inhibitingbone resorption or restenosis in a subject. The invention furtherprovides methods for using an RGD-containing peptide to alter thebinding of a ligand to an α_(V) β₃ integrin receptor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the names and structures of Tic and representative Ticanalogs.

FIG. 2 lists and demonstrates the activity of various RGD peptides ofthe invention. All of the peptides are cyclic except peptides 1, 2, 233and 425, which are linear. Values indicated are mean 50% inhibitoryconcentration (IC50; μM) for the interactions listed across the top ofthe table. IC50 values were determined by linear regression analysis (1to 5 assays), except that numbers in parentheses in the column headed"rat OC attach (FCS)" were obtained from a single experiment using 5 μMpeptide and represent the percent of osteoclasts remaining adherent inthe presence of peptide as compared to osteoclasts remaining adherent inthe absence of peptide. OC, osteoclast; FCS, fetal calf serum; Echi,echistatin; JY, human B lymphocyte-derived JY cell line; Vn,vitronectin; Hep, heparin. Regarding the peptide sequences, "NH2"indicates the presence of an amide group at the C-terminus; dE indicatesthe D-amino acid form of glutamic acid; dF indicates the D-amino acidform of phenylalanine. Other abbreviations are listed in Table 1 (see,also, FIG. 1, for Tic and Tic analogs). SEQ ID NO. is listed forpeptides containing only L-amino acids.

FIG. 3 lists additional RGD peptides of the invention. Underliningindicates the amino acids in the cyclic region of the peptide. SEQ IDNO. is listed for peptides containing only L-amino acids. dPen indicatesthe D-amino acid form of penicillamine. Other abbreviations are listedin Table 1 (see, also, FIG. 1, for Tic and Tic analogs).

FIG. 4 demonstrates the dose response of various RGD peptides in thechick osteoclast cell detachment assay. The IC50 was calculated bydetermining the dose of a peptide that results in the detachment of 50%of the osteoclasts and is shown for each peptide. Echi indicatesechistatin. Peptide TL31 is a negative control.

FIG. 5 demonstrates the selectivity of various peptides in the chicktotal cell detachment assay. A selective peptide produces a curve thatis shifted to the right and has a horizontal shape.

FIG. 6 compares various peptides in both the chick osteoclast detachmentassay (OCs) and chick total cell detachment assay (Total Cells).Peptides showing a greater difference in the ability to detachosteoclasts as compared to total cells are considered more selective.

FIG. 7 demonstrates the effectiveness of various peptides in the ratosteoclast attachment assay using fetal calf serum as substrate.

FIG. 8 demonstrates the selectivity of various peptides in the ratkidney cell attachment assay. Peptides that are selective for bindingα_(V) β₃ integrin, which is not present on normal rat kidney cells, donot affect attachment of the cells, whereas peptides that arenonselective for this integrin show a dose response for inhibition ofcell attachment.

FIG. 9 shows the change in serum ionized calcium resulting from infusionof various peptides at the indicated concentrations.

FIG. 10 demonstrates the effectiveness of various RGD peptides of theinvention in the rat osteoclast attachment assay using osteopontin (OPN)or vitronectin (Vn) as substrate.

FIG. 11 demonstrates the effectiveness of various RGD peptides of theinvention in the rat osteoclast attachment assay using collagen assubstrate .

FIG. 12 demonstrates the effectiveness of various RGD peptides of theinvention in the rat osteoclast attachment assay using fibronectin assubstrate.

FIG. 13 presents the members of the integrin family. The known subunits,the subunit combinations that form the known integrins and the knownligands for these integrins are shown. The RGD specificity of thoseintegrins that bind RGD also are shown. The more recently identified β₆subunit tentatively has been assigned to the α_(V) group. FN,fibronectin; VN, vitronectin; FB, fibrinogen; LM, laminin; vWF, vonWillebrand factor; COLL, collagen; OP, osteopontin; BSP1, bonesialoprotein 1; ICAM-1 and ICAM-2 are intercellular adhesion molecules;C3bi, complement component C3bi; Fn Alt, fibronectin alternativelyspliced domain.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides non-naturally occurring Arg--Gly--Asp(RGD) peptides that can inhibit bone resorption and can inhibit thebinding of an osteoclast to a matrix such as bone (see, for example,Davies et al., J. Cell. Biol. 109:1817 (1989); Horton et al., Expt. CellRes. 135:368 (1990)). RGD peptides of the invention also can bind theα_(V) β₃ integrin and, therefore, can inhibit or promote α_(V) β₃-mediated cell attachment depending on whether they are presented to thecell in a soluble form or bound to a solid substrate, respectively.Since α_(V) β₃ integrins are involved in angiogenesis and in therestenosis that can occur following angioplasty, the RGD peptides of theinvention can be useful for altering angiogenesis and for reducing orinhibiting restenosis in a subject (see Choi et al., J. Vasc. Surg.19:125 (1994); Brooks et al., Cell 79:1157-1164 (1994a); Brooks et al.,Science 264:569 (1994b), each of which is incorporated herein byreference).

As used herein, the term "Arg--Gly--Asp peptide" or "RGD peptide" meansa peptide having at least one Arg--Gly--Asp-containing sequence, asdefined below, which can function as a binding site for an integrin typereceptor (see Table 1 for amino acid codes). Integrin receptors can binda variety of RGD-containing peptides (see, for example, Ruoslahti etal., In Morphoregulatory Molecules (G. M. Edelman et al., eds. 1990);Ruoslahti, J. Clin. Invest. 87:1-5 (1991)).

It is intended that the term "RGD peptide" in its broadest senseincludes a peptide comprising Arg--Gly--Asp or a functional equivalentof Arg--Gly--Asp. For example, an amino acid such as lysine,homoarginine (homoArg), N-methyl arginine or a mimic of these aminoacids is a functional equivalent of arginine. Similarly, mimics of Glyand Asp are functional equivalents of glycine and aspartic acid,respectively. Therefore, a peptide comprising, for example,Lys--Gly--Asp is considered an "RGD peptide" within the meaning of thepresent invention. As used herein, the term "mimic" means an amino acidor an amino acid analog that has the same or a similar functionalcharacteristic of an amino acid. Thus, for example, an arginine analogcan be a mimic of arginine if the analog contains a side chain having apositive charge at physiologic pH, as is characteristic of theguanidinium side chain reactive group of arginine. A peptide mimetic orpeptidomimetic is an organic molecule that retains similar peptide chainpharmacophore groups as are present in the corresponding

                  TABLE 1                                                         ______________________________________                                        Single letter                                                                             Three letter                                                      code        code       Amino acid*.sup.+                                      ______________________________________                                                    Adp        γ-aminoadipic acid                               A           Ala        Alanine                                                            βAla  β-alanine                                                     α-Aba                                                                              α-Amino isobutyric acid                          R           Arg        Arginine                                               N           Asn        Asparagine                                             D           Asp        Aspartic acid                                                      Cha        Cyclohexyl-alanine                                                 Chg        cyclohexyl-glycine                                                 Cit        Citrulline                                             C           Cys        Cysteine                                               Q           Gln        Glutamine                                              E           Glu        Glutamic acid                                          G           Gly        Glycine                                                            t-BuG      tert-butyl Glycine                                     H           His        Histidine                                                          Hpa        Homophenylalanine                                      I           Ile        Isoleucine                                             L           Leu        Leucine                                                            nL         norleucine                                             K           Lys        Lysine                                                 M           Met        Methionine                                                         Mpa        β-Mercaptopropionic acid                                      Msa        β- (methylsulfonyl)                                                      amino!alanine                                                      Mamb       m-(aminomethyl)                                                               benzoic acid                                                       N-Me-R     N-methyl-arginine                                                  Npg        neopentyl-glycine                                                  OMeTic     O-methyl-Tic.sup.+                                                 Orn        Ornithine                                                          p-amino-Phe                                                                              para-amino-                                                                   phenylalanine                                                      Pen        Penicillamine                                                      PgL        phenyl-glycine                                         F           Phe        Phenylalanine                                                      p-Cl-F     para-chloro-                                                                  phenylalanine                                                      p-iodo-Phe para-iodo-phenylalanine                                            Pmc        β,β-pentamethylene                                                  cysteine                                                           Pmp        β,β-pentamethylene-β-                                          mercaptopropionic acid                                 P           Pro        Proline                                                            homoP      homoproline                                                        dhP        dehydroproline                                                     fp         3-phenylproline                                                    Psa        β- (phenylsulfonyl)                                                      amino!alanine                                          S           Ser        Serine                                                             Tca        1,2,3,4-tetrahydro-                                                           β-carboline-3-                                                           carboxylic acid                                                    tetA       β-(1(2)H-tetrazol-                                                       5-yl)-alanine                                          T           Thr        Threonine                                                          Tfsa       β- (trifluoromethyl                                                      sulfonyl)amino!alanine                                 W           Trp        Tryptophan                                             Y           Tyr        Tyrosine                                                           YOMe       O-methyl-tyrosine                                      V           Val        Valine                                                             2-Nal      β-(2-naphthyl) Ala-OH                                         1-Nal      β-(1-naphthyl) Ala-OH                             ______________________________________                                         * -- includes amino acids and analogs thereof                                 .sup.+  -- for Tic and Tic analogs, see FIG. 1                           

peptide. Peptide mimetics also can be functional equivalents ofArg--Gly--Asp.

In one aspect, the RGD peptides of the invention are grouped based onthe pharmacophore. As used herein, the term "pharmacophore" is definedas a particular three-dimensional arrangement of functional groups thatis required for a compound to produce a particular response or have adesired activity. The invention provides, for example, a pharmacophore,abbreviated "RGDNTic," (SEQ ID NO: 31) which is contained within anon-naturally occurring peptide comprising the sequence:

    X.sub.1 X.sub.2 X.sub.3 X.sub.4 GD X.sub.5 X.sub.6 X.sub.7 X.sub.8 (SEQ ID NO: 32)

wherein X₁ is R₁ R₂ N (wherein R₁ is an H or alkyl group and R₂ is an H,alkyl, CH₃ CO, alkyl--CO or phenyl--CO group) or 0 to 10 amino acids,which can be protected by acetylation at the N-terminus; X₂ is 0 or 1amino acid, which, when X₂ is one amino acid, can form a bridge with X₇; X₃ is 0, 1 or 2 amino acids, which, when X₃ is two amino acids, theamino acids can be a pair consisting of Phe--Ala, Ala--Ala, Glu--Ala,Tyr--Ala, Arg--Ala, Ile--Ala, Glu--Pro, Ile--Phe, (2-Nal)--Ala,YOMe--Ala or analogs or mimics thereof; X₄ is a positively charged aminoacid such as Arg, Lys, homoArg, N--Me--Arg or analogs or mimics thereof;X₅ is an amino acid such as Asn, Ser, Thr or mimics or analogs thereof,which can provide a hydrogen bond interaction with an integrin receptor;X₆ is an amino acid such as Tic, Pro, Phe, Ile or analogs or mimicsthereof, which have the characteristics of hydrophobicity and/orconformational constraint; X₇ is a residue capable of forming a bondwith an amino acid of X₂, or with X₃ when X₂ is 0, or with X₄ when X₂and X₃ are 0, to conformationally restrain the peptide; and X₈ is--CONR₃ R₄ (wherein R₃ is an H or alkyl group and R₄ is an H or alkylgroup) or --COOR₅ (wherein R₅ is an H or alkyl group) or 0 to 10 aminoacids, which can be protected as an amide at the C-terminus, and whereinwhen X₅ is serine and X₆ is proline, X₃ is 0 or 2 amino acids. Theabbreviation "RGDNTic" (SEQ ID NO: 31) includes all peptides encompassedby this formula.

The invention also provides, for example, a pharmacophore, abbreviated"RGDDV," (SEQ ID NO: 1) which is contained within a non-naturallyoccurring peptide comprising the sequence:

    X.sub.1 X.sub.2 X.sub.3 X.sub.4 GD X.sub.5 X.sub.6 X.sub.7 X.sub.8 (SEQ ID NO: 33)

wherein X₁ is R₁ R₂ N (wherein R₁ is an H or alkyl group and R₂ is an H,alkyl, CH₃ CO, alkyl--CO or phenyl--CO group) or 0 to 10 amino acids,which can be protected by acetylation at the N-terminus; X₂ is 0 or 1amino acid, which, when X₂ is one amino acid, can form a bridge with X₇; X₃ is 0, 1 or 2 amino acids, which when X₃ is two amino acids, theamino acids can be a pair consisting of Glu--Ala, Tyr--Ala, Arg--Ala,Ile--Gly, Glu--Pro, or a hydrophobic amino acid followed by a secondamino acid such as Ile--Ala, Ile--Phe, (2-Nal)--Ala, Phe--Ala orAla--Ala, or analogs or mimics thereof; X₄ is a positively charged aminoacid such as Arg, Lys, homoArg, N--Me--Arg or analogs or mimics thereof;X₅ is an amino acid such as Asp, Glu, β-(1(2)H-tetrazol-5-yl)-alanine oranalogs or mimics thereof such as the sulfonamide derivatives, Msa, Psaor Tfsa (see Example I.D.), which can form an ionic or similarinteraction with an integrin receptor; X₆ is an amino acid such as Leu,Ile, Val, Thr, Nle or analogs or mimics thereof, which has an aliphaticside chain, or is an aliphatic non-natural amino acid that ishydrophobic such as t-BvG, Cha, Chg, Npg or phenylglycine; X, is aresidue capable of forming a bond with a bridging amino acid of X₂, orwith X₃ when X₂ is 0, or with X₄ when X₂ and X₃ are 0, toconformationally restrain the peptide; and X₈ is --CONR₃ R₄ (wherein R₃is an H or alkyl group and R₄ is an H or alkyl group) or --COOR₅(wherein R₅ is an H or alkyl group) or 0 to 10 amino acids, which can beprotected as an amide at the C-terminus. The abbreviation "RGDDV" (SEQID NO: 1) includes all peptides encompassed by this formula.

In another aspect, the present invention provides RGD-containing cyclicpeptides having the following sequences: G-R-G-D-E-P-D-G (SEQ ID NO:16); R-G-D-N-I-E-NH₂ (SEQ ID NO: 25); G-Pmc-R-G-D-C-A (SEQ ID NO: 34);G-Pen-G-R-G-D-N-Y-C-A (SEQ ID NO: 35); G-Pen-G-E-R-G-D-N-Y-C-A (SEQ IDNO: 36); G-Pen-A-A-R-G-D-V-P-C-A-NH₂ (SEQ ID NO: 37);G-Pen-E-L-R-G-D-G-W-C-NH₂ (SEQ ID NO: 38); G-Pen-G-F-R-G-D-E-P-C-NH₂(SEQ ID NO: 39); K-Pen-G-F-R-G-D-E-P-C-R (SEQ ID NO: 40);K-Pen-G-F-R-G-D-D-P-C-R (SEQ ID NO: 41); Ac-Pen-A-A-R-G-D-Orn-P-C-NH₂(SEQ ID NO: 42); (Mpa)-R-G-D-(YOMe)-(t-BuG)-C-NH₂ (SEQ ID NO:43) and(Mpa)-R-G-D-(Cha)-(t-BuG)-C-NH₂ (SEQ ID NO:44) (underlined amino acidresidues indicate the cyclic region of a peptide). In an additionalaspect, the invention provides a linear peptide having the sequence:Ac-R-G-D-D-V-G-NH₂ (SEQ ID NO: 2). These and other peptides are usefulfor practicing the invention.

As used herein, the term "amino acid" is meant in its broadest sense toinclude naturally occurring proteogenic amino acids and imino acids aswell as non-naturally occurring amino acids and imino acids and analogsand mimics thereof. As used herein, the term "proteogenic" indicatesthat the amino acid can be incorporated into a protein in a cell throughwell known metabolic pathways. In view of this broad definition of anamino acid, one skilled in the art would know that reference herein toan amino acid, unless specifically indicated otherwise, includes, forexample, naturally occurring proteogenic (L)-amino acids, (D)-aminoacids, chemically modified amino acids such as amino acid analogs,naturally occurring non-proteogenic amino acids such as norleucine, andchemically synthesized compounds having properties known in the art tobe characteristic of an amino acid. For example, Tic is aconformationally constrained analog of Phe and a benzo-fused analog ofPro. Tic analogs include, for example, Idc, Dic, Oic, Tca, and 7-OMe-Tic(see FIG. 1 for structure and full names of Tic and Tic analogs). Otherconformationally constrained amino acids are known in the art andinclude, for example, proline derivatives (Chung et al., J. Org. Chem.55:270-275 (1990), which is incorporated herein by reference).

The choice of including an (L)- or a (D)-amino acid in an RGD peptide ofthe present invention depends, in part, on the desired characteristicsof the peptide. For example, the incorporation of one or more (D)-aminoacids can confer increased stability on the peptide in vitro or in vivo,above that provided by conformational constraint, and can allow thepeptide to remain active in the body for an extended period of time. Theincorporation of one or more (D)-amino acids also can increase ordecrease the pharmacological activity of the peptide as determined, forexample, using the assays described in Examples II and III, below, orother in vitro or in vivo assays for determining, for example,osteoclast binding to a matrix or osteoclast activity. The skilledartisan can determine the desirable characteristics required of an RGDpeptide of the invention by taking into consideration, for example, theage and general health of a subject.

Increased stability of a disulfide bonded peptide of the invention alsocan be conferred by incorporating a hydrophobic amino acid residue suchas phenylalanine at position X₁ or X₈ or both X₁ and X₈ in an RGDNTic(SEQ ID NO: 31) or RGDDV (SEQ ID NO: 1) pharmacophore as disclosedherein. The presence of such a residue can stabilize the disulfide bondagainst enzymatic degradation (see Bauer et al., Life Sciences31:1133-1140 (1982)). For example, peptide 220 (see FIG. 2), which iswithin the RGDNTic (SEQ ID NO: 31) class of peptides, contains aphenylalanine residue at position X₁ and exhibits a relatively longhalf-life in serum. Methods for determining pharmacokinetic propertiesof a peptide of the invention such as half-life of a peptide in serumare routine in the art (see, for example, Experimental and SurgicalTechnique in the Rat, 2d ed., chapters 1, 2 and 3 (H. B. Waynforth andP. A. Flecknell; Academic Press Ltd., London 1992); AppliedBiopharmaceutics and Pharmacokinetics, 3d ed., chapters 3-5 and 9 (L.Shargel and A. B. C. Yu; Appelton and Lange, USA 1993), each of which isincorporated herein by reference). In addition, the half-life of apeptide in a subject can be determined, for example, by high performanceliquid chromatography (HPLC) of serum samples collected from the subjectat various times following administration of the peptide. One skilled inthe art would know how to select appropriate elution buffers for HPLCbased on the physico-chemical properties of a particular peptide.

The RGD peptides of the invention can be chemically synthesized using,for example, an automated synthesizer (see Example I). Selectivemodification of a reactive group such as an amino acid side chain or anN- or C-terminal group in a peptide can impart desirable characteristicsto an RGD peptide of the invention. A peptide can be manipulated whilestill attached to the resin following chemical synthesis or aftercleavage from the resin to obtain, for example, an N-terminal modifiedpeptide such as the acetylated peptides disclosed herein (see Example Iand FIGS. 2 and 3). Peptides also can be synthesized containing theC-terminal carboxy group or a C-terminal amide as described in ExampleI. A newly synthesized peptide can be purified using a method such asreverse phase HPLC (RP-HPLC), as described in Example I, or othermethods of separation based on the size or charge of the peptide.Similarly, well known methods such as amino acid sequence analysis ormass spectrometry, as described in Example I, are available forcharacterizing the structure of the RGD peptide.

An RGD peptide of the invention can be constrained by various methods,including, for example, formation of a disulfide bond or a lactam bondbetween X₇ and one of either X₂, X₃ or X₄. Residues capable of forming adisulfide bond include Cys, Pen, Pmp, Pmc and Mpr (βMercaptopropionicacid) (see Table 1). Residues capable of forming a lactam bridge includeAsp, Glu, Lys, Orn, α,β-diaminopropionic acid, γ-amino-adipic acid andm-(aminomethyl)benzoic acid. The RGD peptides disclosed herein cancyclize, for example, via a lactam bond, which can utilize a side chaingroup of X₇ to form a covalent attachment to the N-terminal amine of X₂or X₃ (when X₂ is 0) or X₄ (when X₂ and X₃ are 0). In particular, X₇ ina lactam-bonded peptide can be an Asp or a Glu residue. Conformationalconstraint also can be effected by the choice of a residue such asproline that is incorporated into a peptide or by synthesis of apeptidomimetic having a particular conformation or by any other meansknown to one skilled in the art.

RGD peptides of the invention comprising an RGDNTic (SEQ ID NO: 31)pharmacophore or an RGDDV (SEQ ID NO: 1) pharmacophore are exemplifiedby peptides shown in FIGS. 2 and 3. The minimal elements of the cyclicRGD peptides of the invention include 1) a five amino acid pharmacophoresuch as the pharmacophores abbreviated RGDNTic (SEQ ID NO: 31) or RGDDV(SEQ ID NO: 1) and 2) a means of conformational restraint. Peptide 812(Ac-CRGDS(Tic)C-NH₂ ; SEQ ID NO: 45) from the RGDNTic (SEQ ID NO: 31)pharmacophore group and peptide 386 (RGDDVE-NH₂ ; SEQ ID NO: 3) from theRGDDV (SEQ ID NO: 1) pharmacophore group exemplify RGD peptides thathave these minimally essential elements and demonstrate activity (seeFIG. 2). Peptides are referred to herein by a number, which can be showneither alone or preceded by the letters, TL (see, also, FIG. 2, numberslisted in the left hand column).

Peptides of the RGDNTic (SEQ ID NO: 31) pharmacophore group arecharacterized, in general, as having increased potency, whereas thepeptides of the pharmacophore RGDDV (SEQ ID NO: 1) group generally havehigher receptor selectivity. However, RGD peptides having bothcharacteristics also were identified (see, for example, peptides 750,487, 191 and 159; FIG. 2). As used herein, the term "potency" or"potent" means that a peptide of the invention is active at a relativelylow concentration in the assays described in Examples II and III. Apotent peptide of the invention can be identified, for example, usingthe assays described in Examples II and III.

As used herein, the term "selective" means that a peptide of theinvention preferentially reduces or inhibits the migration or binding toa matrix of cells such as osteoclasts, which are involved in boneresorption, endothelial cells, which are involved in angiogenesis, orsmooth muscle cells, which are involved in neointimal hyperplasia.Selectivity can be due, for example, to the ability of a peptide of theinvention to bind to α_(V) β₃ with greater affinity than it binds toother integrins. Thus, in addition to having selectivity, for example,for osteoclast binding to a matrix, a peptide of the invention also candisplay receptor selectivity and, therefore, more specifically alterreceptor-mediated binding, for example, of an endothelial cell to amatrix or receptor-mediated migration or binding, for example, of asmooth muscle cell to a region of arterial injury. It is recognized thata peptide of the invention can specifically bind more than one integrin,yet still be considered selective because it does not non-specificallybind any integrin. Thus, a peptide such as TL940, which can specificallybind both the α_(V) β₃ and the α_(IIb) β₃ integrin receptors, isconsidered an example of a selective peptide that specifically binds twointegrins.

As used herein, the term "receptor selectivity" means that a peptide ofthe invention can bind to one or more integrin receptors with greateraffinity than it can bind to other receptors. Thus, a peptide of theinvention is selective in that it specifically binds one or a fewintegrins, but does not non-specifically bind any integrin. For example,peptides such as peptides 487, 750, 368, 814, 191, 84 and 159 have a lowIC50 value for α_(V) β₃ binding as compared to α_(V) β₅ binding or α₅ β₁binding (see FIG. 2) and, therefore, are receptor selective for α_(V)β₃. Methods for identifying a selective peptide are described in ExampleII.

Integrins are heterodimeric molecules comprised of an α and a β subunit.The αβ integrin receptor binds to molecules in the extracellular matrix,commonly via the Arg--Gly--Asp sequence present in such molecules.Numerous α and β subtypes have been identified and the combination of aparticular α subtype with a particular β subtype contributes to theligand specificity of the integrin (see FIG. 13). Some integrins canadopt subtle conformational changes specific to a particular cell type,thus providing an additional level of cell type integrin specificity(Chuntharapai et al., Exp. Cell Res. 205:345-352 (1993)).

As used herein, the terms "matrix" and "ligand" are used in the broadestsense to mean a material to which a cell such as an osteoclast, anendothelial cell or a smooth muscle cell can bind to or migrate across.Cells can bind to or migrate across extracellular matrix components suchas fibronectin, vitronectin, laminin and collagen. In addition, it iswell known that osteoclasts, for example, can bind to bone and to bonematrix proteins such as osteopontin and bone sialoprotein.

As disclosed herein, osteoclasts also can bind, for example, to a matrixsuch as a serum-coated microtiter plate or to a plate having attachedthereto an RGD peptide of the invention. In addition, an osteoclast canexpress specific integrin receptors on its cell surface and, therefore,can bind to a matrix component recognized by the receptor. For example,JY cells express α_(V) β₃ integrin receptors and, accordingly, can binda matrix such as vitronectin (see Example II). Vitronectin is a ligandof the α_(V) β₃ receptor.

Vascular cells such as endothelial cells and smooth muscle cells alsocan migrate across and bind to a matrix such as the basement membraneregion of blood vessels. Such interactions of endothelial cells andsmooth muscle cells are involved, for example, in angiogenesis andrestenosis. Angiogenesis, for example, is important for normaldevelopment and wound healing. In addition, however, angiogenesis isrequired for the growth of solid tumors and neovascularization serves asa conduit for metastasis. Anti-α_(V) β₃ antibodies and relativelynon-selective peptide antagonists for α_(V) β₃ integrinreceptor-mediated cell binding suppressed angiogenesis in tumors in thechick chorioallantoic membrane assay (Brooks et al., supra, 1994a;1994b). These results indicate that α_(V) β₃ integrin receptor-mediatedbinding is involved in the vascular cell adhesion to the extracellularmatrix that occurs in angiogenesis.

The α_(V) β₃ integrin receptor also is involved in the migration andbinding of cells to a region of vascular injury such as occurs duringrestenosis. Of the approximately 350,000 angioplasties performed eachyear, restenosis occurs in greater than 30% of the treated patientswithin one year. A peptide that binds the α_(V) β₃ receptor inhibitedthe neointimal hyperplasia that normally occurs following arterialinjury in a rabbit model of atherosclerosis (Choi et al., supra, 1994).In addition, an anti-β₃ antibody reduced clinical restenosis (Topol etal., Lancet 343:881 (1994)). Since β₃ can associate with α_(IIb) as wellas α_(V), more than one integrin can be involved in the vascular cellmigration, binding and proliferation that results in restenosis.

Osteoclasts attach to bone matrix via integrin-type receptors present onthe osteoclast cellular membrane. Integrin receptor-mediated osteoclastbinding, for example, to bone matrix is involved in bone resorption.Since integrin receptor binding is involved in the interaction of cellswith a matrix, a process such as bone resorption, angiogenesis orrestenosis can be reduced or inhibited by reducing or inhibiting, forexample, α_(V) β₃ integrin receptor-mediated binding of a cell such asan osteoclast to bone matrix or of endothelial cell or smooth musclecell to vascular matrix.

Osteoclasts can express an α_(V) β₃ integrin receptor, as well as α_(V)β₁ and α₂ β₁ integrin receptors (Nesbitt et al., J. Biol. Chem.268:16737-16745 (1993)). α_(V) β₁ is a fibronectin receptor. Asdiscussed above, the α_(V) β₃ integrin is a vitronectin receptor, whichcan be involved in bone resorption. For example, a monoclonal antibodyagainst α_(V) β₃ blocked bone resorption in vitro (Horton et al., Exp.Cell Res. 195:368-375 (1991)). The vitronectin receptor present on ratosteoclasts can bind a wide variety of RGD-containing extracellularmatrix and bone proteins in vitro, including osteopontin and bonesialoprotein (Helfrich et al., J. Bone Miner. Res. 7:335-343 (1992);Reinholt et al., Proc. Natl. Acad. Sci. USA 87:4473-4475 (1990);Miyauchi, et al., J. Biol. Chem. 266:20369-20374 (1991)). The α₂ β₁integrin interacts with collagen and laminin and may be involved in thesealing event necessary for resorption to occur (Horton et al., supra,1991). An agent such as the RGD peptides of the invention, which caninhibit ligand binding to these and other integrins present onosteoclasts, is useful for disrupting the binding of osteoclasts to boneand, therefore, reducing or inhibiting the process of bone resorption.

As discussed above, the α_(V) β₃ integrin receptor also is involved inangiogenesis and is required for neovascularization (Brooks et al.,supra , 1994b). Antagonists of α_(V) β₃ can disrupt newly forming bloodvessels but do not affect preexisting vasculature. Systemicadministration of α_(V) β₃ antagonists can inhibit angiogenesisassociated with tumor growth and metastasis and can cause regression ofvarious histologically distinct human tumors (Brooks et al., supra,1994a). As disclosed herein, the claimed peptides, including the RGDNTic(SEQ ID NO: 31) and RGDDV (SEQ ID NO: 1) pharmacophores, are useful foraltering angiogenesis, which can be associated with a pathology such ascancer or retinopathy or with various inflammatory diseases (see, forexample, Blood and Zetter, Biochim. Biophys. Acta 1032:88-118 (1990);Folkman, Sem. Canc. Biol. 3:65-71 (1992)). The peptides of the inventionalso can be useful for promoting neovascularization, for example, byimmobilizing a peptide onto a desirable matrix, thus promoting adhesionand migration of migratory endothelial cells on the matrix. Althoughreference is made herein generally to endothelial cells, it should berecognized that the α_(V) β₃ integrin receptor is expressed by migratoryendothelial cells but not necessarily by all endothelial cells.

The α_(V) β₃ integrin receptor also can be involved in the migration,binding and proliferation of smooth muscle cells at a site of arterialinjury, resulting in a pathology characterized, for example, by theneointimal hyperplasia associated with restenosis of an artery followingangioplasty. Antagonists of the α_(V) β₃ integrin receptor, including,for example, an anti-α_(V) β₃ antibody and the peptide G(Pen)GRGDSPCA,(SEQ ID NO: 46), can inhibit neointimal hyperplasia (Choi et al., supra,1994). As disclosed herein, the RGD peptides of the invention can beuseful for reducing or inhibiting restenosis and, in addition, canprovide the advantage of having greater potency or selectivity or bothas compared to G(Pen GRGDSPCA (SEQ ID NO: 46).

A β₃ -containing integrin receptor such as the platelet α_(IIb) β₃integrin receptor also can be involved in restenosis (Topol et al.,supra, 1994). Thus, a peptide of the invention such as TL200 or TL697,both of which selectively bind the α_(IIb) β₃ integrin receptor, can beused alone or in combination with a peptide that selectively binds theα_(V) β₃ integrin receptor to reduce or inhibit restenosis in a subject.In addition, a peptide of the invention such as TL940, which binds boththe α_(V) β₃ and α_(IIb) β₃ integrin receptors, can be administeredalone to a subject in order to reduce or inhibit restenosis in thesubject.

The invention provides a method of reducing or inhibiting boneresorption, angiogenesis or restenosis in a subject, comprisingadministering an RGD peptide as disclosed herein. As used herein, theterms "reducing" and "inhibiting" have their common meanings. The termsare used together, here, to avoid any ambiguity as to the extent towhich an RGD peptide of the invention acts. It is recognized that an RGDpeptide can decrease, for example, the level of bone resorption below alevel that is detectable using a particular assay. In this situation,one would be unable to determine whether the rate of bone resorption wasreduced to a lower level or inhibited such that no resorption wasoccurring. The use of these terms together precludes the need todistinguish such events.

The peptides of the invention also can alter α_(V) β₃ integrinreceptor-mediated binding. As used herein, the term "alter" is used inits broadest sense to indicate an increase or decrease in binding. Forexample, when present in solution, the peptides of the invention candecrease α_(V) β₃ binding to a ligand by competing with the ligand forthe integrin receptor. A peptide of the invention also can be attachedto a solid substrate, which can increase binding of the α_(V) β₃receptor to the substrate. Thus, a peptide of the invention can beuseful, for example, for altering angiogenesis in a tissue. For example,an endothelial cell can be contacted with a peptide of the invention,which can inhibit the binding of an endothelial cell to a matrix,thereby reducing or inhibiting angiogenesis. Alternatively, a peptide ofthe invention can be immobilized onto a matrix such that endothelialcells can bind to the matrix, thereby increasing angiogenesis in oraround the matrix.

Integrin-ligand binding can be inhibited and binding can be disrupted bysynthetic peptides comprising the RGD sequence (see, for example, U.S.Pat. No. 4,614,517 issued Sep. 30, 1986; U.S. Pat. No. 4,792,525 issuedDec. 20, 1988; U.S. Pat. No. 4,988,621 issued Jan. 29, 1991, each ofwhich is incorporated herein by reference). RGD-containing peptides havebeen shown to block osteoclast adhesion in vitro (Horton et al., J. BoneMiner. Res. 8:239-247 (1993)). Echistatin is a natural RGD-containingprotein that is isolated from snake venom. Echistatin binds toosteoclasts in a nonselective manner and inhibits bone resorption invitro (Sato et al., J. Cell Biol. 111:1713-1723 (1990); EP 437,367) andin vivo (Fisher et al., Endocrinology 132:1411-1413 (1993).

The potency and selectivity of an RGD peptide toward an integrinreceptor can be improved by restricting the number of conformations thepeptide can adopt (Pierschbacher and Ruoslahti, J. Biol. Chem.262:17294-17298 (1987); WO 89/05150). Conformational restraint of apeptide can be induced by various means including, for example,cyclization. The concept of using cyclic RGD peptides, modeled afterechistatin, to inhibit osteoclast attachment to bone has been broadlydescribed (EP 437,367), and osteoclast attachment inhibiting activitiesof several cyclic RGD peptides have been reported (Bertolini et al., In13th Ann. Mtg. Am. Soc. Bone Miner. Res., abstract 252 (1991); Horton etal., supra, 1993).

In one aspect, an RGD peptide useful as a therapeutic for unwanted boneresorption, angiogenesis or restenosis is receptor selective intargeting only the one or few integrins involved in bone resorption,angiogenesis or restenosis, respectively, and not others. As a result,an RGD peptide of the invention can preferentially inhibit, for example,the binding of osteoclasts, as compared to other cell types, to bone. Inanother aspect, a therapeutically useful RGD peptide is potent and,therefore, effective at a relatively low dose. A peptide having bothpotency and selectivity is particularly useful for reducing orinhibiting bone resorption. In comparison, a peptide such as TL940 thatis potent but can bind both α_(V) β₃ and α_(IIb) β₃ receptors can beuseful for reducing or inhibiting restenosis. The present inventionprovides RGD peptides having one or more of these characteristics andprovides methods for evaluating RGD peptides to identify selective andpotent peptides and methods of using the RGD peptides of the inventionto inhibit bone resorption, angiogenesis or restenosis.

The present invention also provides a series of in vitro and in vivoassays useful for screening a panel of peptides in order to identifypeptides that can reduce or inhibit bone resorption or that can alterintegrin binding such as α_(V) β₃ integrin receptor-mediated binding.Such peptides can include, for example, peptides within thepharmacophore groups exemplified herein or peptides within otherpharmacophore groups. The series of assays disclosed herein isparticularly useful for determining the selectivity and potency of apeptide for reducing or inhibiting bone resorption. The selectivity of apeptide for an integrin receptor involved in osteoclast binding to bonecan be determined, for example, using the integrin receptor assays andcell attachment and detachment assays described herein. Use ofcell-based assays in conjunction with receptor-based assays isinformative because the receptor ELISAs provide useful informationregarding the binding characteristics of a particular peptide toward aparticular integrin and because the cell-based assays confirm that thosecharacteristics are relevant to the α_(V) β₃ receptor or to boneresorption.

A peptide that selectively binds an integrin receptor involved inosteoclast binding to bone and that causes detachment of osteoclasts,but not other cell types, can be identified as a selective peptide andcan be further examined for its potency by comparing the IC50 value ofthe peptide with other peptides. Thus, the disclosed series of assaysprovides a means for identifying a potent and/or selective peptide,which can reduce or inhibit bone resorption or alter α_(V) β₃ binding.

A potent or selective RGD peptide can be administered to a subject suchas a human in order to reduce or inhibit bone resorption, angiogenesisor restenosis in the subject. With regard to bone resorption, thesubject has a pathology that is characterized by an undesirable balanceof bone resorption as compared to bone formation, resulting in excessivebone resorption. An elderly person suffering from osteoporosis is anexample of a subject having such a pathology. Other relevant pathologiesinclude, for example, Paget's disease, osteoclastoma and thepathological loss of bone that occurs in an astronaut during a spacemission or in a person exposed to a minimal gravity environment.Furthermore, bone loss can occur in a bone that is immobilized in acast, in a subject restricted to a prolonged period of bed rest and in asubject undergoing kidney dialysis. Administration of an RGD peptide ofthe invention can reduce or inhibit the rate of bone resorption in eachof these subjects. Thus, the peptides of the invention are particularlyuseful as medicaments, wherein administration of such a peptide to asubject can decrease the amount of bone loss, angiogenesis or restenosisin the subject.

An α_(V) β₃ selective peptide of the invention also can be useful in apathology such as cancer, provided the cancer cell, for example,expresses the α_(V) β₃ integrin. Administration of a peptide of theinvention to a subject having such a pathology can inhibit attachment ofthe α_(V) β₃ integrin-containing cell to extracellular matrix proteinsor other relevant ligands involved in tumor cell metastasis (Nip et al.,J. Clin. Invest. 90:1406-1413 (1992); Gladson and Cheresh, J. Clin.Invest. 88:1924-1932 (1991), each of which is incorporated herein byreference). Peptides of the invention that can inhibit α_(V) β₃ integrinbinding to a ligand and, thus, can be effective in treating a pathologysuch as cancer are evaluated using methods known in the art anddescribed, for example, by Hardan et al., Intl. J. Canc. 55:1023-1028(1993); Murthy et al., Clin. Expt. Metast. 10:39-47 (1992); and Komazawaet al., Clin. Expt. Metast. 11:482-491 (1993), each of which isincorporated herein by reference.

The α_(V) β₃ integrin also is involved in the binding of endothelialcells to von Willebrand factor (VWF). This binding is necessary formaintaining the integrity of the blood vessel wall and permitting newblood vessel formation and blood vessel wall repair (Denis et al., Blood82: 3622-3630 (1993)). α_(V) β₃ receptor selective peptides can be usedto strengthen attachment of endothelial cells to vessel walls or toinhibit the interaction of an endothelial cell and VWF. Similarly,angiogenesis depends on interactions between endothelial cells and α_(V)β₃ integrins (Brooks et al., supra, 1994a). Pathologies such as diabeticretinopathy, rheumatoid arthritis and cancer can result due topathologies affecting these interactions. Thus, peptides that areselective for binding to the α_(V) β₃ integrin can bind α_(V) β₃ andblock its binding to a ligand. Such selective binding by a peptide ofthe invention can provide a means of treating an α_(V) β₃ -relatedpathology, including, for example, the restenosis that occurs in anarterial wall following angioplasty or the angiogenesis that occursduring growth and metastasis of a tumor.

Peptides selective for binding to α_(V) β₃ also can affect binding tothe α_(IIb) β₃ integrin, which is involved in platelet aggregation. Forexample, the peptide RGD(YOMe)RE-NH₂ (SEQ ID NO: 47) (TL940; see FIG. 2)has an IC50 value of 0.0042 μM in an α_(V) β₃ ELISA and is highly potentat inhibiting platelet aggregation (IC50=0.82 μM). Thus, an α_(V) β₃selective peptide also can be useful, for example, for treating apathology such as restenosis, which involves the α_(IIb) β₃ receptor(Choi et al., supra, 1994; Topol et al., supra, 1994). Similarly,peptide ligands for the α_(IIb) β₃ receptor can be useful for inhibitingbone resorption or for reducing or inhibiting restenosis. Assays usefulfor evaluating the reactivity of a peptide with α_(IIb) β₃, includingwhether the peptide can inhibit platelet aggregation, are known in theart and described herein and, for example, in International PublicationNo. WO91/15515, published Oct. 17, 1991, which is incorporated herein byreference.

The in vitro assays disclosed in Example II also can be used to identifyreceptor selective peptides of the invention that have various specificin vitro and in vivo utilities. For example, a receptor selectivepeptide of the invention can be immobilized on a supporting matrix suchas a natural or synthetic surface. Such a peptide coated surface can beuseful for separating in vitro a population of cells that express aspecific receptor from cells that do not express the receptor. Forexample, a peptide that selectively binds α_(V) β₅ but not α₅ β₁ orα_(V) β₃ can be used to isolate a population of normal articularchondrocytes, which primarily express α_(V) β₅ integrin receptors, fromcells that express α_(V) β₃ and/or α₅ β₁ integrins. A peptide coatedmatrix can be produced by immobilizing the peptide on the matrix, forexample, through a covalent or non-covalent bond or other interactionsuch that the peptide is associated with the matrix.

Various receptor selective peptides are disclosed herein or can beidentified using the methods in Example II. For example, peptides 159,487 and 750 have higher affinity for α_(V) β₃ than for α_(V) β₅ and α₅β₁. Thus, peptides 159, 487 and 750 can bind with greater affinity to acell such as an osteoclast, which expresses α_(V) β₃ as compared to acell that expresses α_(V) β₅ and α₅ β₁ receptors. In comparison,peptides 100, 825, 390 and 310 are more selective toward α₅ β₁ thantoward α_(V) β₅ and can be useful to isolate a population of cellsexpressing an α₅ β₁ receptor.

The receptor selectivity of a peptide of the invention also can be usedadvantageously in vivo. For example, a peptide coated matrix asdescribed above can be implanted into a subject in order to facilitatelocalization of cells expressing a specific integrin receptor at theimplant site. For example, peptide 214 as described above can be usefulfor inducing the localization of osteoclasts at a site of bonehypertrophy as occurs in a pathology, such as osteopetrosis or senileankylosing hyperostosis of the spine, or can be useful for inducingangiogenesis, for example, when performing natural or artificialvascular grafts or dermal replacements. Thus, a peptide of the inventioncan be useful for increasing angiogenesis where such angiogenesis isotherwise insufficient. In addition, peptides 100, 825, 390 and 310, asdescribed above, can be useful for inducing α₅ β₁ -mediated fibronectinmatrix assembly, for example, in a wound. If desired, the peptides ofthe invention can be administered to a subject in soluble form and,therefore, can be useful for preventing α₅ β₁ -mediated fibronectinmatrix assembly in a wound in order to prevent, for example, excessivescarring. Thus, the receptor selectivity of the disclosed peptides asshown, for example, in FIG. 2 defines various utilities for the specificpeptides.

An RGD peptide of the invention also can be used to reduce or inhibitangiogenesis under conditions where the occurrence or amount ofangiogenesis is inappropriate. As used herein, the term "inappropriate"when used in reference to angiogenesis means an unwanted or excessivegrowth of blood vessels. For example, tumor growth is dependent, inpart, on angiogenesis, which facilitates nutrient transport to theproliferating tumor cells. Such angiogenesis is unwanted from a clinicalperspective and, therefore, inappropriate as defined herein. Inaddition, a pathology can be associated with excessive angiogenesis, inwhich a case a peptide of the invention can be useful for reducing orinhibiting angiogenesis. Thus, an objective of the present invention isto provide an RGD peptide of the invention to a subject having apathology characterized, in part, by inappropriate angiogenesis for thepurpose of reducing or inhibiting such angiogenesis.

The invention provides pharmaceutical compositions comprising an RGDpeptide of the invention, which can reduce or inhibit bone resorption,angiogenesis or restenosis or alter α_(V) β₃ binding, and apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are well known in the art and include, for example, aqueoussolutions such as physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, oils such as olive oil or injectableorganic esters. A peptide of the invention also can be encapsulatedwithin a biodegradable or non-biodegradable polymer.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize an RGD peptideof the invention or to increase the absorption of the peptide. Suchphysiologically acceptable compounds include, for example,carbohydrates, such as glucose, sucrose or dextrans, antioxidants, suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins or other stabilizers or excipients. One skilled in the artwould know that the choice of a pharmaceutically acceptable carrier,including a physiologically acceptable compound, depends, for example,on the route of administration of the RGD peptide and on the particularphysico-chemical characteristics of the specific peptide.

A pharmaceutical composition comprising an RGD peptide of the inventioncan be useful for ameliorating the severity of a pathologycharacterized, in part, by the involvement of α_(V) β₃ integrinreceptor-mediated binding of a cell. Such pathologies are manifested,for example, by an undesirable level or occurrence of bone resorption,angiogenesis, neointimal hyperplasia or restenosis in a subject, whereinthe undesirable level can be undesirably high or undesirably low. Forexample, a pathology such as osteoporosis or cancer is characterized, inpart, by undesirably high levels of bone resorption or angiogenesis,respectively. In these cases, an objective of the present invention isto decrease α_(V) β₃ integrin receptor-mediated binding of a cell to amatrix. In addition, a pathology can be characterized, in part, by anundesirably low level, for example, of bone resorption and, therefore,an objective would be to increase osteoclast binding to the bone matrixby coating the matrix with an RGD peptide of the invention. Thus, theterm "pathology" is used broadly herein to include any conditioncharacterized, in part, by an undesirable level of bone resorption,angiogenesis or restenosis. The present invention provides compositionsand methods for ameliorating the severity of such a pathology. As usedherein, the term "ameliorate" has its commonly known meaning of "makebetter," "improve," "alleviate" or "relieve."

A peptide of the invention can be administered, for example, to asubject suffering from a pathology such as osteoporosis, which ischaracterized, in part, by bone resorption, or a pathology such ascancer, which is characterized, in part, by angiogenesis of the growingtumor, wherein such administration can ameliorate the severity of thepathology. Similarly, an RGD peptide of the invention can beadministered to a subject treated by angioplasty in order to amelioratethe severity of restenosis, which can be a pathologic response toangioplasty.

A peptide of the invention also can be administered to a subjectexperiencing, for example, abnormal bone growth, in which case the RGDpeptide can be used to increase the α_(V) β₃ integrin receptor-mediatedbinding of osteoclasts to the bone by coating the bone matrix with anRGD peptide of the invention such that the peptide is immobilized on thematrix. In addition, a peptide of the invention that is selective for α₅β₁ binding can be useful, for example, for treating osteoporosis byimmobilizing such peptides to a bone surface in order to promoteadhesion of osteoblasts, which are involved in bone formation, to thebone surface. In view of the present disclosure, other uses for the RGDpeptides of the invention would be known to those skilled in the art andare described, for example, by Grzesik and Robey, J. Bone Min. Res.9:487 (1994). Furthermore, it is recognized that a peptide of theinvention can be useful in a soluble form, in which case it caninterfere with the binding of an α_(V) β₃ -containing cell with amatrix, or can be useful when immobilized on a matrix, in which case itcan mediate the binding of an α_(V) β₃ -containing cell to the coatedmatrix.

One skilled in the art would know that a pharmaceutical compositioncomprising an RGD peptide of the invention can be administered to asubject having a pathology characterized, in part, by bone resorption,angiogenesis or restenosis by various routes including, for example,orally or parenterally, such as intravenously, intramuscularly,subcutaneously or intraperitoneally or by passive or facilitatedabsorption through the skin using, for example, a skin patch ortransdermal iontophoresis, respectively. Furthermore, the compositioncan be administered by injection, intubation or topically, the latter ofwhich can be passive, for example, by direct application of an ointmentor solution, or active, for example, using a nasal spray or inhalant. Apharmaceutical composition comprising an RGD peptide of the inventionalso can be administered as a topical spray, in which case one componentof the composition can be an appropriate propellant. The pharmaceuticalcomposition also can be incorporated, if desired, into liposomes ormicrospheres or can be microencapsulated in other polymer matrices(Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton, Fla.1984), which is incorporated herein by reference). Liposomes, forexample, which consist of phospholipids or other lipids, are nontoxic,physiologically acceptable and metabolizable carriers that arerelatively simple to make and administer.

In order to ameliorate the severity of a pathology characterized, inpart, by bone resorption, angiogenesis or restenosis in a subject or toalter α_(V) β₃ or other integrin binding, an RGD peptide of theinvention must be administered in a therapeutically effective amount,which is about 0.01 to 100 mg/kg body weight/day. As used herein, theterm "therapeutically effective amount" means an amount of a peptidethat can ameliorate the severity of a pathology characterized, in part,by bone resorption, angiogenesis or restenosis in a subject.

A therapeutically effective amount of a peptide can be determined usingmethods known to those in the art, including the methods described inExamples II and III, below. A therapeutically effective amount of apeptide can be administered to a subject as a single dose, either as abolus or by infusion over a relatively short period of time, or can beadministered using a fractionated treatment protocol, in which themultiple doses are administered over a more prolonged period of time.One skilled in the art would know that the concentration of an RGDpeptide of the invention required to obtain a therapeutically effectiveamount in a subject depends on many factors including the age andgeneral health of the subject as well as the route of administration andthe number of treatments to be administered. In view of these factors,the skilled artisan would adjust the particular dose so as to obtain atherapeutically effective amount for ameliorating the severity of apathology characterized, in part, by bone resorption, angiogenesis orrestenosis in a subject.

An RGD peptide of the invention is useful for reducing or inhibitingosteoclast binding to a matrix in vitro. As generally described in U.S.Pat. Nos. 4,614,517, 4,792,525 and 4,988,621, supra, RGD-containingpeptides in solution bind to integrins that are expressed on the surfaceof a cell and either prevent the cell from attaching to a matrix oreffect detachment of the cell from a matrix. In addition, whenRGD-containing peptides are coated onto a surface, a cell that expressesan integrin reactive with the peptide can bind to the surface. Thus, thepeptides of the invention can be used to alter the ability of a cellexpressing an integrin to attach to a solid matrix such as a tissueculture plate, a prosthetic device or an artificial extracellular matrix(see, for example, WO 90/06767, which is incorporated herein byreference). Since peptides of the invention also can demonstratereceptor selectivity, they are particularly useful for inhibiting thebinding of a cell expressing the integrin to such a matrix.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I Synthesis of RGD Peptides

This example provides methods for chemically synthesizing the RGDpeptides of the invention.

A. Synthesis of Cyclic Peptides Having Disulfide Bonds

Peptides were synthesized by the solid-phase method utilizing anautomated synthesizer (Applied Biosystems, Inc. Model 431A) (see Stewardand Young, In Solid Phases Peptide Synthesis, 2nd ed. (Pierce ChemicalCo., Rockford, Ill., 1984), which is incorporated herein by reference).Peptides having a C-terminal amide were synthesized usingp-methylbenzhydrylamine (pMBHA) resin. Peptides having a C-terminal acidwere synthesized using chloromethylated resin. Peptides having anN-terminal acetyl group were acetylated using a mixture of aceticanhydride (20 eq) and diisopropylethylamine (20 eq) inN-methylpyrrolidone.

N-terminal tertbutyloxycarbonyl (Boc) protection was employed for allamino acids. Boc--Arg(Tos)--OH, Boc--Asp(OcHx)--OH,Boc--Cys(4-MeBzl)--OH, Boc--Gly--OH, Boc--Hpa--OH, Boc--Lys(CZ)--OH,Boc--Pen(4-MeBzl)--OH, Boc--(D)Pen(4-MeBzl)--OH, Boc--Pro--OH,Boc--Tic--OH, Boc-7-OMe--Tic and Boc--Tyr(Me)--OH were purchased fromBachem Inc. (Torrance, Calif.). The following compounds were synthesizedusing the methods described in the indicated references: Boc--Idc--OH(Sancy, In Organic Syntheses 63:160-170 (1984)); Boc--Dic--OH (Martin etal., EP 432,695); Boc--OiC--OH (Vincent et al., Tetr. Lett. 23:1677-1680(1982)); and Boc--Tca--OH (Yabe et al., Chem. Pharm. Bull. 26:993-997(1978), each of which is incorporated herein by reference). Mamb(m-(aminomethyl) benzoic acid) was prepared as described by Jackson etal. (J. Am. Chem. Soc. 116:3220-3230 (1994), which is incorporatedherein by reference) and was incorporated into a peptide using an oximeresin. Dicyclohexylcarbodiimide and hydroxybenzyltriazole were used inthe coupling reactions. The extent of the reactions was monitored usingthe standard ninhydrin test.

Following synthesis, the peptides were removed from the resin anddeprotected by adding anhydrous hydrogen fluoride (HF; 10 ml/g ofresin-bound peptide) containing anisole (1 ml/g) at 0° C. for 60 min.The HF was removed by evaporation and the residue was washed withanhydrous ether. The crude peptides were extracted with water or 15%aqueous acetic acid and the aqueous fractions were combined andlyophilized. Peptides were purified and characterized as describedbelow.

The crude acyclic peptide was dissolved in 0.1M ammonium bicarbonate(0.5 mg/ml) and stirred uncovered. The course of the reaction wasmonitored by HPLC. After cyclization was complete (several hours toseveral days), the solution was filtered and the peptides were purifiedand characterized as described below.

B. Synthesis of Cyclic Peptides Having Lactam Bridges

Cyclic peptides having a lactam bridge were synthesized as outlinedherein. The protected peptide resin was synthesized using the PMBHAresin. The lactam bridge was formed while the peptide was on the resinusing the method described by Felix et al., Int. J. Pept. Prot. Res.31:231 (1988) and by Felix et al., Int. J. Pept. Prot. Res. 32:441(1988), each of which is incorporated herein by reference.

Essentially, the method of Felix et al. uses N.sup.α --Boc-amino acidstogether with 9-fluorenylmethyl ester (OFm) side-chain protection. Aspand Glu were introduced using Boc--Asp(OFm)--OH and Boc--Glu(OFm)--OH.After coupling the final amino acid, OFm protecting groups wereselectively removed by treating the peptide resin with 50% piperidine indimethylformamide for 1 hr. The peptide resin was washed with 3×40 mldichloromethane and mixed with a 6-fold excess of BOP reagent(benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphoniumhexafluorophosphate) in the presence of an 8-fold excess ofdiisopropylamine for 5 hr. Coupling reactions were repeated until theresin gave the negative ninhydrin test.

After the cyclization reaction was complete, peptides were removed fromthe resin and deprotected using anhydrous HF (10 ml/g of resin-boundpeptide) containing anisole (1 ml/g) at 0° C. for 60 min. The HF wasremoved by evaporation and the residue was washed with anhydrous ether.The crude peptides were extracted with water or 15% aqueous acetic acidand the aqueous fractions were combined and lyophilized and purified andcharacterized as described below.

C. Purification and Characterization of the Peptides

The crude peptides were purified via preparative RP-HPLC on a C₁₈ silicagel column "WATERS DELTA-PAK" Column, 15 μm, 300A, 47×300 mm) elutingwith a linear acetonitrile gradient (0-30%) with a constantconcentration of trifluoroacetic acid (TFA; 0.1%, v/v) over 30 min at aflow rate of 40 ml/min. The purified peptides were analyzed byanalytical RP-HPLC using a C-18 column (Vydac, 5 μm, 300A, 4.5×250 mm).The purified peptides were recovered by lyophilization of the HPLCfractions and were at least 95% pure. For analytical HPLC, a binarysolvent system, water containing 0.1% TFA and acetonitrile containing0.1% TFA as the organic modifier, was used. The solvent programsinvolved linear gradients as follows: (1) 10-45% acetonitrile over 35min with a flow rate of 1.5 ml/min and (2) 0-70% acetonitrile over 30min with flow rate of 1.5 ml/min.

In some cases, the peptide was adjusted to a neutral pH and potassiumferricyanide was added to the TFA peptide to minimize polymerizationthat might result due to the presence of the reducing agent. Thepotassium ferricyanide was removed by ion exchange chromatography andthe peptides were lyophilized. The presence of thiol reducing agents,which indicates the need to add potassium ferricyanide, can be detectedusing Ellman's test (Arch. Biochem. Biophys. 82: 70 (1959), which isincorporated herein by reference).

To confirm the correct amino acid sequences as shown in FIGS. 2 and 3were synthesized, the peptides were characterized by fast atombombardment mass spectroscopy and by amino acid analysis. Amino acidanalysis was performed on a Pickering Labs-Trione amino acid analyzerthat was equipped with spectra-physics UV detector. Hydrolysis ofpeptide samples for amino acid analysis was performed on 1 mg sampleswith 1 ml constant oiling 6N HCl. Samples were degassed, sealed undervacuum nd heated for 24 hr at 110° C.

D. Synthesis of β- (methylsulfonyl)amino!alanine

This example describes the synthesis of β (methylsulfonyl)amino!alanine(Msa). Psa and Tfsa were synthesized according to the same procedure,except that methanesulfonylchloride was replaced withphenylsulfonylchloride for synthesis of Psa and withtrifluoromethylsulfonylchloride for synthesis of Tfsa.

Msa was synthesized using the general procedure of Krchnak et al.,Collection Czechoslov. Chem. Comm. 44:2161-2164 (1979), which isincorporated herein by reference). To a stirred solution of N.sup.α-tertbutyloxy carbonyl-α,β-diaminopropionic acid (700 mg, 3.43 mmol) in1 N NaOH (34 ml) was added a solution of methanesulfonyl chloride (530μl, 6.86 mmol) in acetone (30 ml).

After 1 hr, the reaction was extracted 2× with ether and acidified to pH2 using 1 N HCl. The aqueous phase was extracted 3× with ethyl acetateand the combined organic phases were dried (MgSO₄) and concentrated toproduce an oil. Msa was purified by silica gel chromatography usingethyl acetate/ethanol/acetic acid (95.8:4:0.2) as eluant. 300 mg Msa wasobtained and was used in the standard solid phase peptide synthesisprotocol. ¹ H-NMR analysis: (MeOH, D₄ ; 300 MHz) 4.2 ppm; (m, 1H).3.3-3.6 ppm; (m, 2H). 2.95 ppm; (s, 3H). 1.4 ppm (s, 9H).

EXAMPLE II RGD Peptide Activity In Vitro

This example describes a series of assay useful for determining theactivity of an RGD peptide for inhibiting bone resorption, includinginhibiting the binding of an osteoclast to a matrix in vitro.

A. Preparation of Assay Reagents 1. Isolation of Human Vitronectin

Human plasma was clotted by adjusting the sample to 20 mM CaCl₂, thenstirring for 1 hr at room temperature (RT), followed by 2 hr at 4° C.The plasma was fractionated by centrifugation at 3000 rpm for 15 min("SORVALL" centrifuge with GS3 rotor) and the supernatant was retained.

The supernatant was adjusted to 1 mM phenylmethylsulfonyl fluoride(PMSF)/5 mM EDTA. A column containing 75 ml "SEPHAROSE CL-4B" (beadedagarose) (Pharmacia; Piscataway N.J.) was equilibrated using five columnvolumes (5 vol) phosphate buffered saline (PBS), the supernatant wasadded and the flow-through fraction was collected. The flow-throughfraction was applied to a column containing 50 ml heparin- "SEPHAROSE"(beaded agarose) which was made by swelling heparin-"SEPHAROSE CL-6B"(beaded agarose) (Pharmacia) with PBS for 5 min in a sintered glassfunnel, then washing with PBS for 15 min. The heparin-"SEPHAROSE"(beaded agarose) was poured into a column, washed with 3 vol Buffer B (5mM EDTA, 2M NaCl, 10 mM sodium phosphate, pH 7.7) and equilibrated with5 vol PBS. The dye-colored flow-through fraction was collected, solidurea was added to a final concentration of 8M and the sample wasincubated with stirring for 2 hr at RT.

The heparin "SEPHAROSE" (beaded agarose) column was washed with at least3 vol Buffer C (5 mM EDTA, 8M urea, 10 mM sodium phosphate, pH 7.7, 10mM 2-mercaptoethanol, 2M NaCl) and equilibrated with 5 vol Buffer A (5mM EDTA, 8M urea, 10 mM sodium phosphate, pH 7.7). The flow-throughfraction from above was loaded onto the column. The column was washedwith 3 vol Buffer A, followed by 3 vol Buffer A containing 140 mM NaCland by 1 vol Buffer A containing 140 mM NaCl/10 mM 2-mercaptoethanol.After 2 hr, the column was washed with 3 vol of the same final buffer.

Vitronectin (Vn) was eluted using Buffer D Buffer A containing 0.5MNaCl). The fractions were evaluated by 8% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under non-reducingconditions. The presence of Vn was confirmed by identifying thecharacteristic 65 kilodalton (kDa) and 75 kDa bands. In some instances,Vn was confirmed by western blot analysis using a specific anti-Vnantibody (8Eb; Hayman et al., Proc. Natl. Acad. Sci., USA 80:4003-4007(1983), which is incorporated herein by reference). Western blotanalysis was performed using a standard method as described, forexample, by Sambrook et al., Molecular Cloning: A laboratory manual(Cold Spring Harbor Laboratory Press 1989), which is incorporated hereinby reference.

2. Isolation of Human Fibronectin

Frozen human plasma was thawed at RT, then adjusted to 100 μM PMSF. Theplasma was fractionated by centrifugation at 3000 rpm for 15 min in a"SORVALL" centrifuge (GS3 rotor) and the supernatant was collected.

A column containing 300-500 ml "SEPHAROSE-4B" (beaded agarose) wasequilibrated with 1 l Buffer E (8 g KCl, 8 g KH₂ PO₄, 320 g NaCl, 86.8 gNaH₂ PO₄ per liter). The plasma supernatant was added to the column andthe flow-through was collected from the time the yellow-colored plasmaenters the "SEPHAROSE" (beaded agarose) until the time the eluate becameclear. Two vol Buffer E were added to the column as a wash buffer to"push" the plasma through the column.

A column containing 50 ml gelatin "SEPHAROSE-4B" (beaded agarose) wasprepared in a Kontes column and equilibrated with 500 ml Buffer E. Theflow-through from the "SEPHAROSE-4B" (beaded agarose) column was loadedat a rate of 2 ml/min or slower. The column was washed with 500-1000 mlBuffer E or until OD₂₈₀ was below 0.10.

Three to five vol Buffer F (0.05M Tris, 4.5M urea, pH 7.0) were used toelute plasma fibronectin (Fn). One-quarter column vol fractions werecollected and evaluated by 8% SDS-PAGE under reducing conditions. Thepresence of Fn was confirmed by identifying the characteristic bandmigrating at approximately 210-250 kDa. In some cases, the presence ofFn was confirmed by western blot analysis using a specific anti-Fnantibody (3E1; Pierschbacher et al., Cell 26:259-267 (1981), which isincorporated herein by reference).

Peak fractions containing Fn were pooled and the optical density wasmeasured. If necessary, the pooled fraction was diluted until an OD₂₈₀reading corresponding to 1 mg/ml Fn was obtained. The sample then wasdialyzed against PBS and stored at -20° C. in PBS in a non-frost freefreezer.

3. Preparation of 110 kDa Fibronectin Fragment

Fibronectin was purified as described above and dialyzed against 3changes of 100 vol Buffer G (0.05M Tris, 0.3M NaCl, pH 8.3-8.5). Theurea concentration decreased to below 0.1M.

Chymotrypsin was added at a concentration of 0.001 mg chymotrypsin/mgFn. The digestion mixture was incubated overnight at 4° C. with gentlerocking. The presence of complete digestion was confirmed by SDS-PAGEanalysis of pre- and post-digestion samples. The digested Fn sample wasdialyzed against 2 changes of 100 vol Buffer H (0.1M NaHCO₃, 0.5M NaCl,pH 8.0). The pH of the dialyzed digest was between pH 8-9.

4. Purification of Human α_(V) β₅ Vitronectin Receptor (VnR)

Human placenta was homogenized and washed on ice in 225 ml ofTris-buffered saline (TBS; pH 7.4-7.5; 4° C.), then fractionated bycentrifugation at 3000 rpm for 15 min at 4° C. The supernatant wasdiscarded and the pellet was extracted with 225 ml TBS containing 100 mMoctyl-glucopyranoside (OG). Extraction was performed at 4° C., on ice,for 30 min, with stirring every 10 min. The extract was fractionated bycentrifugation at 3000 rpm for 20 min and the supernatant was collected.The supernatant, which contained the α_(V) β₅ Vn receptor (VnR), wasadjusted to 3 mM CaCl₂.

Two tandem Kontes (Fisher Scientific) columns were prepared using equalvolumes of "SEPHAROSE" (beaded agarose) 4B or 6B. Both columns wereequilibrated with 10 vol cold (4° C.) Buffer I (1X TBS, pH 7.4, 50 mMOG, 3 mM CaCl₂, 20 μg/ml PMSF). One column contained Vn (Vn column) andwas prepared as follows. Vn was dialyzed against 2 to 3 changes ofcoupling buffer (0.1M NaHCO₃, 0.5M NaCl, pH 8.3). CnBr-"SEPHAROSE"(beaded agarose) was swelled in 1 mM HCl, washed with coupling buffer,then combined with Vn. Coupling proceeded for 2 hr at RT or overnight at4° C., followed by blocking with 1M ethanolamine or 0.2M glycine (pH8.0) for 2 hr at RT.

The filtered, calcium supplemented placental extract was loaded onto the"SEPHAROSE" (beaded agarose) column, then run through the Vn column at4° C. The flow-through was collected in a polypropylene container fordisposal. The Vn column was washed with 5 vol cold (4° C.) Buffer I,allowed to equilibrate to RT, then washed with another 5 vol Buffer I atRT. The column was slowly eluted with 3 vol Buffer J (1X TBS, pH 7.4, 50mM OG, 100 μg/ml GRGDSP (SEQ ID NO: 4) peptide, 20 μg/ml PMSF).Fractions were collected and evaluated by 8% SDS-PAGE under non-reducingconditions. Fractions showing bands migrating at the characteristic 150,145 and 90 kDa molecular mass of the VnR were pooled, dialyzed againstTBS (pH 7.5) containing 50 mM OG/3 mM CaCl₂ and concentrated using an"AMICON YM 30" filter.

5. Purification of Human α₅ β₁ Fibronectin Receptor (FnR)

Fibronectin receptor (FnR) was purified from 100 mMoctyl-glucopyranoside-extracted human placenta. The procedure forpurifying the FnR was the same as described for purifying the VnRthrough the initial "SEPHAROSE" (beaded agarose) chromatography step.Then, for purifying the FnR, the Sepharose 4B column flow-through wasadjusted to 3 mM Mn²⁺ and the resulting solution was fractionated usingaffinity chromatography on columns containing a GRGDSPK (SEQ ID NO: 5)peptide and the 110 kDa fibronectin fragment. Column washes and elutionswere the same as described for purifying VnR, except that MnCl₂ ratherthan CaCl₂ was included in the wash buffer.

Briefly, an unmodified "SEPHAROSE 4B" (beaded agarose) column wasequilibrated with 5 vol cold (4° C.) Buffer K (TBS, pH 7.5, 50 mMoctylglucoside, 3 mM CaCl₂). The flow through was collected and adjustedto a final concentration of 3 mM CaCl₂. A GRGDSPK-"SEPHAROSE" (beadedagarose) affinity column (20-40 ml) was prepared using the method forpreparing the Vn column, above, except that lyophilized GRGDSPK (SEQ IDNO: 5) was suspended in the coupling buffer and the sample was notdialyzed. The column was equilibrated with 200-400 ml Buffer K andplacental extract was applied. The flow-through fraction was collectedand adjusted to 6 mM MnCl₂.

The 110 kDa Fn fragment-"SEPHAROSE" (beaded agarose) column was preparedas described for the Vn column. The column was preequilibrated with 5vol cold Buffer L (TBS, pH 7.5, 50 mM octylglucoside, 3 mM MnCl₂), theextract was applied and the flow-through fraction was collected. Thecolumn was washed with 5 vol cold Buffer L, allowed to come to RT (about30 min), then another 5 vol Buffer L at RT was added. When the OD₂₈₀ ofthe flow through was below 0.02, FnR was eluted using 3 vol Buffer M(TBS, pH 7.5, 50 mM octylglucoside, 10 mM EDTA) at RT and one-quartercolumn volume fractions were collected. The fractions were evaluated by8% SDS-PAGE under non-reducing conditions and fractions exhibiting bandsmigrating at the characteristic 155 kDa and 110 kDa molecular masses ofFnR (Hemler, Ann. Rev. Immunol. 8:365-400 (1990), which is incorporatedherein by reference) were pooled, dialyzed into TBS (pH 7.5) containing50 mM octylglucoside/3 mM MnCl₂ and concentrated using an "AMICON YM 30"filter.

6. Purification of Human α_(V) β₃ Vitronectin Receptor

The α_(V) β₃ receptor was purified from octyl-glucopyranoside-extractedhuman placenta prepared as described in Example II.4., above. Two tandemKontes (Fischer Scientific) columns were prepared using equal volumes of"SEPHAROSE" (beaded agarose) 4B or 6B. The peptide VTRGDVFTK (SEQ ID NO:6) was coupled to one column using the method described in ExampleII.5., above (peptide column). The other column was not modified (plaincolumn). Both columns were equilibrated with 5 vol cold (4° C.) Buffer N(1X TBS, 50 mM octylglucoside, 3 mM MnCl₂).

The placental extract was applied to the plain column and the flowthrough was applied to the peptide column. Following application of theflow through, the peptide column was washed with 10-15 vol Buffer N;half the washes were conducted at 4° C. and half were at RT. Elution waswith 4 vol Buffer 0 (1X TBS, 50 mM octylglucoside, 10 mM EDTA) and 1/4vol fractions were collected. The fractions were evaluated on 8%SDS-PAGE under non-reducing conditions. Fractions containing bandsmigrating at the characteristic apparent 150 kDa and 90 kDa molecularmasses of the α_(V) β₃ receptor were pooled and dialyzed overnightagainst 20 mM Tris (pH 8.0), 34.5 mM octylglucoside, 2 mM CaCl₂, and 2mM MgCl₂.

The dialyzed receptor was loaded onto a "MONO-Q" quaternary amine column(HR 5/51; Pharmacia). The pumps were washed with a gradient of Buffer A₁and B₁ before sample loading. Buffer A₁ is 20 mM Tris, pH 8.0, 34.5 mMoctylglucoside, 2 mM CaCl₂, 2 mM MgCl₂ ; Buffer B₁ is Buffer A₁containing 1M NaCl. Both buffers were filtered and degassed prior toadding octylglucoside.

The sample was loaded onto the sample loop (valve 1.1, 1 ml/min, 0% B)and injected onto the column (valve 1.2). One ml fractions werecollected at 0.05 or 0.1 absorbance units. The appropriate fractionswere run on SDS-PAGE and fractions containing α_(V) β₃ integrin werepooled and dialyzed against TBS and 50 mM octylglucoside. The integrinwas concentrated using an "AMICON" YM 30 filter.

7. Preparation of Recombinant Human Osteopontin

Human osteopontin was produced in CHO cells using standard recombinantDNA methods (see, for example, Sambrook et al., supra, 1989). PlasmidpBS/OP (Young et al., Genomics 7:491-502 (1990), which is incorporatedherein by reference), which contains the cDNA sequence encoding humanosteopontin, was digested with XhoI, then treated with DNA polymerase(Klenow) and dinucleotide triphosphates to generate blunt ends. Theplasmid was digested with XbaI and the approximately 1493 bp fragmentcontaining the osteopontin cDNA insert was purified by electrophoresison a 0.8% agarose gel.

Plasmid PRC/CMV (Invitrogen; San Diego Calif.) was digested with BstXIand treated with DNA polymerase (Klenow) and dinucleotide triphosphatesto generate blunt ends. The plasmid was digested with XbaI and thelinearized vector was purified on a 0.8% agarose gel. The 1493 bposteopontin cDNA was ligated with the linearized vector to produce thehuman osteopontin expression vector, hOP/PRC/CMV, which exhibited a 1900bp band following digestion with BamHI and electrophoresis in a 0.9%agarose gel.

Approximately 10 μg purified hOP/PRC/CMV DNA was cotransfected with 1 μgpSV2-dhfr into 80% confluent CHO cells (ATCC CRL 9096) (Subramani etal., Mol. Cell. Biol. 1:854-864 (1981), which is incorporated herein byreference). Positive transfectants were grown in the presence ofmethotrexate and selected on the basis of dhfr expression. Recombinanthuman osteopontin was purified from the CHO cell medium byimmunoaffinity using rabbit anti-human osteopontin antibody (Telios; SanDiego Calif.) coupled to CNBr "SEPHAROSE 4B" (Pharmacia). Purifiedosteopontin was analyzed by SDS-PAGE and migrated with an apparentmolecular mass of about 72-76 kDa under non-reducing conditions.

B. In Vitro Assays for Determining RGD Peptide Activity

1. Chick Cell Detachment Assay

Peptides were evaluated for their ability to detach osteoclasts from amatrix consisting of serum-coated microtiter plates. In prescreeningassays, 5 μM of the various RGD peptides was used to identify RGDpeptides that released at least 60% of the osteoclasts from the matrix.

For RGD peptides that were in the active range as determined by theprescreen, the concentration of active RGD peptide that resulted in therelease of 50% of the osteoclasts (IC50) was determined (see FIG. 4). Ingeneral, RGD peptides having an IC50 of 3.0 μM or less were selected forfurther examination using the assays described below. However, somepeptides having IC50 values greater than 3.0 μM also were analyzed.

Osteoclasts were isolated from embryonic chicks using a modification ofthe methods of Boyde et al., Br. Dent. J. 156:216 (1984) and Chambers etal., Endocrinology 116:234 (1985), each of which is incorporated hereinby reference. Briefly, chick long bones (femur and tibia) were dissectedfree of soft tissue and minced in prewarmed minimum essential medium(MEM, pH 7.2; Irvine Scientific; Irvine, Calif.) with Earl's salts,supplemented with 10% heat-inactivated fetal calf serum (FCS) and 200 mMglutamine and containing 100 μg/ml streptomycin and 100 IU/mlpenicillin. Cells were removed from bone fragments by vigorous shakingin a capped centrifuge tube. Large particulate material was allowed tosettle prior to removal of the cell suspension. After repeated agitationof the cells with a large transfer pipette, 100 μl aliquots of cellswere added in triplicate to Corning 96 well microtiter plates that hadbeen preincubated for 1 hr with 100 μl of MEM/FCS media.

The cells were incubated for 90 min at 37° C. in an atmospherecontaining 10% CO₂. Following incubation, the cell layers were carefullywashed 2× with 200 μl sterile PBS, then 100 μl of fresh MEM/FCS wasadded. Peptides were diluted in the same medium and added to each well.The cells were incubated overnight as described above. After 20 to 24hr, media were aspirated and the cells were fixed in 10% formalin andwashed 2× with distilled water to remove nonadherent cells and debris.Cell layers were stained for. 5 min using 0.1% crystal violet andmultinucleated osteoclasts were counted by light microscopy.

FIG. 2 shows the IC50 values obtained for various RGD peptides and forthe naturally occurring protein, echistatin (ECHI), using the chickosteoclast attachment assay (column designated "chick OC detach (FCS)").FIG. 4 shows the dose response curves for selected RGD peptides.

The chick cell detachment assay also was used to characterize theselectivity of an RGD peptide. The chick cell preparation contains aheterogeneous population of cells, including osteoclasts, osteoblasts,fibroblasts, monocytes and other cell types. A nonselective peptide caninhibit the attachment of these various cell types to the matrix in thisassay, whereas a selective peptide according to this invention primarilywill inhibit osteoclasts binding.

RGD peptides that are selective for releasing osteoclasts, but not theother cell types, from the matrix were identified by extracting thecrystal violet stain from fixed cells and comparing the amount ofstaining in a control well (no peptide) with the amount of stainingfollowing incubation of the cells with an RGD peptide. Crystal violetwas extracted using 20% acetic acid and the optical density of theresulting solution was measured in 96 well microtiter plates byspectrophotometry at 562 nm.

Selectivity of a peptide can be characterized as shown in FIG. 5. Apeptide that was relatively nonselective indiscriminately detached anincreasing number of cells with increasing dose, yielding a decliningpercent attachment dose-response curve. In contrast, more highlyselective peptides detached primarily osteoclasts and had no additionaleffect once osteoclasts were detached from the matrix until highconcentrations of the peptides were attained. Such a selective peptideyielded a curve that was shifted to the right relative to thenonselective peptides; i.e., much higher concentrations of selectivepeptides were needed to detach cells other than osteoclasts. As shown inFIG. 5, peptides 386 and 20, for example, yielded a curve that isshifted to the right, which indicates that peptides 386 and 20 causedselective detachment of osteoclasts as compared to other cell types.FIG. 6 provides a different visualization of the cell type selectivitydata and shows that increasing concentrations of a peptide canselectively detach osteoclasts, as evidenced by the sharp decline in thepercent of attached cells, as compared to other cell types, which arenot readily detached.

2. Rat Osteoclast Bone Resorption Assay

RGD peptides that were active in the osteoclast detachment assay wereevaluated for their ability to inhibit bone resorption. The boneresorption assay further characterizes the potency of an RGD peptide.

Bovine bone femurs were obtained from Pel-Freeze (Rogers, Ariz.). Oneinch transverse slices were cut using a hand saw and bone plugs were cuton a drill press equipped with a 1/4 inch plug cutter. 600 μm thick boneslices were made using a Buehler "ISOMET 2000" diamond saw (Irvine,Calif.). Slices were stored in a 48 well plate containing 10% ethanol.

To remove debris, each slice was sonicated ("FISHER 50 SONICDISMEMBRATOR" in in ethanol and transferred to a sterile 96 well plate.Alternatively, the bovine bone slices were stored and sonicated in PBS,then placed in individual wells of a chamber slide (Lab-Tek; Nunc;Naperville Ill.). 100 μl of a medium containing 10% FCS and 2%streptomycin/penicillin/glutamine was added to the wells and the plateswere incubated at 37° C. for 4 hr.

In addition, tibias, femurs and humeruses were removed from ten ortwelve two-day old rat pups (Harlan Sprague, Indianapolis, Ind.) andsoft tissue and periosteum was removed. The bones were placed in a petridish on ice and 3 ml (10 rat pups) or 4 ml (12 rat pups) of medium asabove was added. The bones were minced, transferred to a 15 ml conicaltube and vigorously shaken for 2 min. The suspension was poured througha 100 μm nylon mesh, which then was washed with 2-5 ml of medium.

In some cases, after transfer to the 15 ml conical tube, the suspensionwas triturated vigorously for 2 min instead of shaken. The suspensionwas poured through the 100 μm nylon mesh and 2-5 ml medium was added tothe bone fragments remaining in the tube. After 2 min trituration, thesuspension was filtered as before.

The cell suspension was agitated using a sterile transfer pipette,medium was removed from the wells containing bovine bone slices and 100μl of the cell suspension (about 4×10⁴ cells) was added to each wellcontaining a bone slice. Peptides were diluted in PBS, 5 μl of thepeptide solution was added to each well and the plates were incubatedfor 18 to 22 hr at 37° C. in an atmosphere containing 10% CO₂.

When chamber slides were used, the cell suspensions were added and thesamples were incubated for 30 min at 37° C., 12.5% CO₂. The cells thenwere gently removed from the slide chamber, leaving the bone slices intheir gasket on the slide. Tweezers were used to remove the bone slices,which were washed in warm medium for 8 sec. The slices were placed intothe wells of a 12 well tissue culture plate (Corning; Corning N.Y.)containing various peptide dilutions and incubated for 18-22 hr at 37°C., 12.5% CO₂.

Following incubation, the supernatant was aspirated and 0.5% trypsin (1×trypsin-EDTA solution; Irvine Scientific; Santa Ana, Calif.) was addedto each well containing a bovine bone slice. After 5 or 10 min, thewells were aspirated and 1% "TRITON X-100" (polyoxyethylene ether)(Sigma; St. Louis, Mo.) was added to dissolve the cells. The plate wasshaken for 1 hr, then each slice was placed in deionized water,sonicated, dehydrated in 70% ethanol for 5 min and air dried. The boneslices were stained with Millipore-filtered 1% toluidine blue in 1%sodium borate for 30 sec, then washed 3× with deionized water. "WHATMAN"No. 1 filter paper was used to blot the sample, then the slices were airdried. The number of pits per slice was determined by light microscopy("LABPHOT-2" microscope, Nikon; Melville, N.Y.). The IC50 value wasobtained by comparing the number of pits on a bone slice that wastreated with a peptide as compared to an untreated slice. The control isset at 100%, which was at least 15 pits/slice. Active peptides reducethe number of pits/slice.

The results of the bone resorption assay are shown in FIG. 2 (columndesignated "Rat Bone (Pits)"). A low IC50 value in this assay indicatedthat a relatively low concentration of the RGD peptide inhibited atleast one essential step in bone resorption. RGD peptides that wereactive in this assay were considered particularly promising candidatesfor use as a pharmacological agent, since this assay utilizes amammalian system and measures the ability of a peptide to effect thedesired end point of inhibiting bone resorption.

3. Rat Osteoclast Attachment Assay

To determine the effect of a peptide on the attachment of ratosteoclasts in vitro, 100 μl aliquots of the rat pup bone cellsuspension described in Example II.B.2., above, were added in triplicateto 96 well "MAXISORP" plates coated with specific substrates or withFCS. Substrate coated plates were used to determine the ability ofpeptides to inhibit attachment or induce detachment of an osteoclastfrom a ligand such as vitronectin, fibronectin, osteopontin or collagenI (Collaborative Research), which are ligands present in bone.Substrates were added to 96 well plates (50 μl of a 5-10 μg/ml solutionin sodium carbonate, pH 9.5) and incubated for 2 hr at 37° C. For FCScontrol wells, 100 μl 10% FCS was added. Wells were aspirated, thenwashed 2× with PBS and blocked with 1% bovine serum albumin in PBS for 2hr at 37° C. Wells were aspirated and washed again 2× with PBS. Cellswere seeded in duplicate at 4×10⁴ cells/well.

Immediately following addition of the cells, peptides were added to eachwell and the plates were incubated for 1 hr at 37° C., 10% CO₂.Alternatively, peptide was added before cell addition. PBS was aspiratedfrom the precoated wells and 10 μl of each peptide dilution was added,then 100 μl of cell suspension (about 4×10⁴ cells/well) was added. Theplate incubated for 1 hr at 37° C., 10% CO₂.

Following incubation, the medium and nonadherent cells were removed byaspiration from each well. Cells were fixed in acetone/citrate, washed2× with distilled water, then stained with 0.1% crystal violet for 5min. Adherent osteoclasts were counted by light microscopy. The resultsof these experiments are shown in FIG. 7 (FCS), FIG. 10 (osteopontin andvitronectin), FIG. 11 (collagen) and FIG. 12 (fibronectin).

Adherent osteoclasts also were counted using immunocytochemistry.Following the 1 hr incubation of the cells with the peptides, 200 μlblocking solution was added to each well and incubated for 1 hr at RT(blocking solution is 10% normal goat serum in 1X TBSC (1X TBSC is 0.3%casein in TBS). Immediately after aspiration, 50 μl of 0.1 μg/ml primaryantibody was added to the appropriate wells and incubated 1 hr at RT ona shaker. The primary antibody was F-11 (Pharmingen; San Diego Calif.)diluted to 0.1 mg/ml with TBSC adjusted to 1:66 of normal goat serum.The cells then were rinsed in TBST (10X TBST is 200 μl "TRITON X-100"(polyoxyethylene ether) and 100 ml of 10X TBS) and the wells were filledwith TBST. The wells were allowed to stand for 20 min on the shaker.

Immediately after aspiration of TBST, 50 μl 1:300 biotinylated goatanti-mouse Ab (Vector; Temecula Calif.) was added to each well. Antibodydilution was in TBSC as above. Incubation and rinsing were as describedabove for the F-11 antibody, except that the incubation time was 30 minand the shaking rinse time was to 15 min.

After aspiration of the second rinse, an inhibiting solution (10 ml 50%methanol, 400 μl hydrogen peroxide, 200 μl sodium azide) was added toeach well and allowed to stand for 15 min at RT. The wells were rinsedwith water, then filled with water and allowed to stand for 5 min. Afteraspiration, this rinsing procedure was repeated with 1X TBSABC (90 ml10X TBS, 20.22 g NaCl, volume adjusted to 1 liter); the second rinse wasallowed to stand for 10 min before aspiration.

ABC solution (1:50 avidin solution and 1:50 biotin solution (Vector) in1X TBSABC; freshly made) was added to each well and incubated for 30 minat RT. The wells were washed with TBSABC and shaken at low speed for 20min on the shaker. After aspiration, bound antibody was detected usingVIP Substrate (Vector) according to the manufacturer's recommendations.After aspiration and air-drying, adherent osteoclasts were counted bylight microscopy.

Mammalian osteoclasts primarily express α_(V) β₃, α₂ β₁ and α_(V) β₁integrins. Human MG63 osteosarcoma cells and normal rat kidney (NRK)cells contain α_(V) β₅, α₂ β₁ and α₅ β₁ surface receptors. Forcomparison to osteoclasts, these cells are considered normal withrespect to the expression of multiple integrins capable of attachment tomore than one ligand. Because MG63 and NRK cells lack high expression ofα_(V) β₃, the range of activity of peptides on these cells reflects therange of selectivity.

MG63 and NRK cell lines were purchased from ATTC (Rockville Md.).Cultures were maintained in DMEM containing 10% FCS. For attachmentassays, cells were trypsinized, resuspended in serum-free DMEM andseeded in duplicate at 4×10⁴ cells/well in 96 well microtiter platesthat had been precoated with 10% FCS. Peptides were added and the cellswere incubated for 1 hr at 37° C., 5% CO₂. Following incubation, celllayers were washed gently with warm DMEM, fixed in 10% formalin for 25min, then stained with 0.1% crystal violet for 5 min. Followingstaining, the stain was extracted with 20% acetic acid and the OD₅₆₂ ofthe extracted stain was determined. The OD values obtained for wellscontaining peptide were compared to control wells, which representmaximum binding. The results of these experiments on NRK cells are shownin FIG. 8 and in FIG. 2 for both cell types.

Selectivity of a peptide can be determined by comparing the results ofFIGS. 7 and 8 (see, also FIG. 2). Peptides that were highly active (lowIC50) both on osteoclast attachment and on MG63 or NRK attachment areconsidered relatively nonselective. In contrast, peptides that werehighly active on osteoclast attachment but were less active on MG63 orNRK attachment are considered selective (TL191, TL386, TL159, TL487 andTL750).

4. JY Cell Attachment Assay

JY cells are human-derived B lymphocyte cells that express the α_(V) β₃VnR, which is the receptor that most likely is involved in osteoclastbinding to bone (Stupack et al., Expt. Cell Res. 203:443-448 (1992),which is incorporated herein by reference). JY cells do not express theα_(V) β₅ VnR. RGD peptides were analyzed for their ability to inhibitthe attachment of JY cells to vitronectin-coated plates.

Binding in the JY attachment assay is particularly relevant as a measureof the potency of a peptide, since JY cells are human cells and theα_(V) β₃ VnR present on JY cells likely is the primary receptor involvedin osteoclast attachment to bone. In chick osteoclasts, both α_(V) β₃and α_(V) β₅ are expressed and likely involved in attachment. Inaddition, the JY attachment assay, when compared with thereceptor-specific enzyme-linked immunosorbent assays (ELISAs) describedbelow, demonstrates the selectivity of RGD peptides for inhibitingbinding due to the α_(V) β₃ VnR.

JY cells were grown in suspension in RPMI media (Irvine Scientific;Irvine, Calif.) supplemented with 10% heat inactivated fetal bovineserum (FBS) and 200 mM glutamine and containing 100 IU/ml penicillin and100 μg/ml streptomycin. A 96 well ELISA plate (Linbro, Titertek) wascoated with 100 μl/well of 5 μg/ml human Vn in sodium carbonate (pH 9.5)and incubated overnight at 4° C. The plate was rinsed 3× with PBS andblocked by adding 100 μl/well of 2 mg/ml BSA in PBS and incubating for 1hr. Following blocking, the wells were washed 2× with PBS.

While the wells were being blocked, the JY cells were rinsed 2× withPBS, then resuspended to 10×10⁵ cells/ml in serum-free RPMI containing 2mg/ml BSA, 200 mM glutamine, 100 IU/ml penicillin, 100 μg/mlstreptomycin and 0.5 mM MnCl₂. At this time, 50 ng/ml phorbol myristateacetate (PMA; Sigma) was added to the cell suspension. PMA stimulatesthe cells and allows them to bind Vn.

RGD peptides were diluted in the serum-free RPMI and 50 μl of eachdilution was added to duplicate wells. 50 μl of the JY cell suspensionwas added to each well and the plate was incubated at 37° C. for 45 min,7% CO₂. Nonadherent cells were gently aspirated from the wells and thewells were rinsed 1× with PBS. Cells were fixed in 10% formalin in PBSfor 10 min, then stained with 0.1% toluidine blue for 1 hr. The platewas rinsed with distilled water and the cells were solubilized using 1%SDS. The amount of staining was determined by spectrophotometry at 595nm and the IC50 was calculated as the concentration of an RGD peptidethat inhibited JY cell binding to Vn by 50% (see FIG. 2, columndesignated "JY attach (α_(V) β₃ /Vn)").

5. Effect of RGD Peptides on Binding to α_(V) β₅,α₅ β₁ and α_(V) β₃Integrin Receptors--ELISA

Peptide binding to purified human α_(V) β₅ and α_(V) β₃, which areVnR's, and to α₅ β₁, which is a FnR, was determined using competitiveELISA assays as described below. If desired, an α_(IIb) β₃ ELISA can beperformed as described in WO91/15515.

a. α_(V) β₅ ELISA

For the α_(V) β₅ assays, Vn was immobilized and the binding ofsolubilized α_(V) β₅ in the presence of various concentrations of apeptide analogue was detected using a mouse monoclonal antibody to humanα_(V) (clone VNR 147; Gibco; Gaithersburg, Md.). Binding of the antibodywas visualized using a second antibody conjugated to horseradishperoxidase (BioRad; Richmond, Calif.).

Microtiter plates were coated at RT by adding 100 μl of 10 μg/ml humanVn in 0.05M sodium carbonate buffer (pH 9.6). The plates were incubatedovernight at 4° C., then washed 3× with TBS containing 0.05% "TWEEN 20"(polyoxyethylenesorbitan monolaurate) ("TWEEN 20"(polyoxyethylenesorbitan monolaurate)). RGD peptides were diluted in TBScontaining 20 mM OG, 2 mM CaCl₂ and 2 mM MgCl₂ and 50 μl was added toeach well at 10-fold serial dilutions. Human α_(V) β₅ was diluted in thesame buffer and 50 μl was added to each well. Similar results wereobserved when the receptor was added prior to addition of the peptide.

The plates were incubated for 3 hr at RT, then washed with 300 μlTBS/"TWEEN 20" (polyoxyethylenesorbitan monolaurate) buffer. Boundreceptor was incubated with 100 μl of the monoclonal anti-α_(V) antibody(diluted in TBS/"TWEEN 20" (polyoxyethylenesorbitan monolaurate) for 2hr, then the wells were washed three times with TBS/"TWEEN 20"(polyoxyethylenesorbitan monolaurate). 100 μl of a 1:3000 dilution ofaffinity purified goat anti-mouse IgG conjugated to horseradishperoxidase was added to each well and the plates were incubatedovernight at 4° C. Following incubation, the plates were washed 3× withTBS/"TWEEN 20" (polyoxyethylenesorbitan monolaurate) and 100 μl of thesubstrate mixture (10 mg O-phenylenediamine in 25 ml 0.1M citratephosphate buffer, pH 5.0, 6 μl 30% H₂ O₂) was added to the wells. Thereaction was allowed to develop for 10-15 min in the dark, then thedevelopment reaction was stopped by adding 50 μl of 4 N H₂ SO₄ to eachwell.

The amount of reaction product was quantitated by spectrophotometry at490 nm. The IC50 was determined by computer using a Molecular Devicesmicroplate reading spectrophotometer and "SOFTMAX" 2.2 software using4-parameter analysis. Nonspecific binding of antibody was measured inwells that did not contain receptor and this value was subtracted fromtotal binding measured in wells containing receptor to yield thespecific binding.

b. α₅ β₁ ELISA

Peptide binding to purified human α₅ β1 (FnR) also was determined usinga competitive ELISA. In these assays, Fn was immobilized to the wellsand the binding of solubilized FnR in the presence of variousconcentrations of a peptide analogue was determined using a mousemonoclonal antibody to human β₁ (clone P4C10; Gibco) and specificbinding was visualized using a second antibody.

Microtiter wells were coated at RT by adding 100 μl of 2 μg/ml human Fnin TBS and incubating the plates overnight at 4° C. Followingincubation, the plates were washed 3× with TBS/"TWEEN 20"(polyoxyethylenesorbitan monolaurate). RGD peptides were diluted in TBScontaining 20 mM OG and 2 mM MnCl₂ and 50 μl was added to wells at10-fold serial dilutions. FnR was diluted in the same buffer and 50 μlwas added to each well.

The plates were incubated for 3 hr at RT and washed with 300 μlTBS/"TWEEN 20" (polyoxyethylenesorbitan monolaurate). Bound receptor wasincubated with 100 μl of the monoclonal anti-β₁ antibody for 2 hr, thenwashed 3× with TBS/"TWEEN 20" (polyoxyethylenesorbitan monolaurate). 100μl of a 1:3000 fold dilution of affinity purified goat anti-mouse IgGconjugated to horseradish peroxidase was added to each well andincubated overnight at 4° C. The amount of bound second antibody wasdetermined as described above.

c. α_(V) β₃ ELISA

Peptide binding to purified α_(V) β₃ receptor was determined by using acompetitive ELISA, in which α_(V) β₃ receptor was immobilized and thebinding of solubilized VN, in the presence of various concentrations ofpeptide analogue, was detected using a monoclonal mouse IgG anti-VNantibody and a labelled anti-mouse IgG conjugate. A monoclonal anti-VNantibody can be obtained using routine methods (see, for example, Harlowand Lane, Antibodies: A laboratory manual (Cold Spring Harbor LaboratoryPress 1988), which is incorporated herein by reference).

96 well microtiter plates were coated overnight at RT with 100 μl/wellof α_(V) β₃ receptor (0.5 μg/ml in 1X TBS, 4 mM octylglucoside, 1 mMCaCl₂). The wells were blocked with 200 μl/well of 1% BSA in 1X TBS, 1mM CaCl₂ for at least 30 min. The plates were washed 2× with 1X TBS, 1mM CaCl₂. 50 μl peptide in 1X TBS, 1 mM CaCl₂, 0.05% "TWEEN 20"(polyoxyethylenesorbitan monolaurate) was added in 10-fold serialdilutions. 50 μl of Vn in the same buffer then was added to each well.

The plates were incubated for 3 hr at RT, then washed 3× with 1X TBS,0.05% "TWEEN 20" (polyoxyethylenesorbitan monolaurate). 100 μl of 8E6(Telios) diluted to 1:6000 in 1X TBS, 0.05% "TWEEN 20"(polyoxyethylenesorbitan monolaurate) was added to the wells and theplates were incubated for 1 hr at RT. Plates were washed 3× with 1X TBS,0.05% "TWEEN 20" (polyoxyethylenesorbitan monolaurate). Affinitypurified goat anti-mouse IgG conjugated to horseradish peroxidase (100μl/well; BioRad; Hercules Calif.) was added to each well and incubatedfor 1 hr at RT. Plates were washed 3× with 1X TBS, 0.05% "TWEEN 20"(polyoxyethylenesorbitan monolaurate). 100 μl/well of substrate mixture(10 mg O-phenylenediamine in 25 ml 0.1M citrate-phosphate buffer, pH5.0, 6 μl 30% H₂ O₂) was added to the plates and allowed to develop. Thedevelopment process was stopped by adding 50 μl to 4 N H₂ SO₄ to eachwell. The plates were read at 490 nm and the data analyzed by fourparameter fit.

d. Analysis of ELISA results

The IC50 values for various RGD peptides were calculated by determiningthe concentration of a peptide that inhibited binding of Vn to α_(V) β₅or α_(V) β₃ or of Fn to α₅ β₁ by 50%. The results of these assays areshown in FIG. 2 (columns designated "Receptor Assays").

Comparison of the IC50 values generated using the JY cell assay and theα_(V) β₃ ELISA, which measure binding of the α_(V) β₃ receptor involvedin osteoclast binding to bone, with the α_(V) β₅ and α₅ β₁receptor-specific ELISAs, which represent receptors that likely are notinvolved in osteoclast attachment, provides insight into the receptorselectivity of the peptides. For example, a peptide such as peptide 368,which has an IC50 value for the α_(V) β₃ ELISA or the α_(V) β₃ VnRpresent on JY cells that is significantly lower than its IC50 value forthe α_(V) β₅ and α₅ β₁ receptors indicates that peptide 368 is aselective peptide.

In summary, the above series of assays are useful for determining thepotency and selectivity of a peptide. Examples of particularly receptorselective peptides include 386, 814, 191, 228, 186, 368, 138, 448, 159,487 and 750. Examples of particularly potent peptides include 373, 202,196, 206, 197, 199, 183, 832, 812, 20, 437, 441, 429, 110, 214, 108, 431and 751. Examples of peptides that are both potent and selective include191, 368, 159, 487 and 750.

6. Effect of RGD Peptides on Platelet Aggregation

This example provides a method for performing a platelet aggregationassay, which is a measure of peptide binding to the platelet α_(IIb) β₃integrin receptor.

Platelet-rich plasma (PRP) was prepared fresh from heparinized wholeblood. 0.6 ml PRP was placed in a 1.5 ml microcentrifuge tube andcentrifuged for 4 min at 14,000×g. Following centrifugation, 0.5 mlsupernatant (platelet poor plasma; PPP) was transferred to a 1 mlsilicon coated aggregometer tube (Chrono-Log Corp.; Havertown Pa.) andplaced in the PPP slot of a Chrono-Log Model 400VS aggregometer (seeTschopp et al., Thromb. Haem. 72:119-124 (1994), which is incorporatedherein by reference).

Platelet concentration of the PRP sample was determined using a cellcounter and the sample was diluted with homologous PPP to obtain 300,000platelets/μl. 0.5 ml of the diluted PRP was placed in an aggregometertube with a stir bar and incubated for 5 min at 37° C. without stirring.The tube was transferred to the PRP slot in the aggregometer and thesample was stirred at 1200 rpm for 5-10 min at 37° C. Peptides weredissolved in physiological saline and added at various concentrations toa PRP sample in the aggregometer. Saline, alone, was added for controlreactions.

Platelet aggregation was initiated by adding adenosine diphosphate (ADP)to a concentration of 10 μM. Platelet aggregation was measured in 2 mmunits from baseline (before ADP addition) to response at two min fromthe time of ADP addition or maximal response (if before 2 min),whichever is larger. Peptide inhibitory activity was determined bycomparing aggregation in the presence and absence of peptide and theconcentration (μM) of peptide that inhibited platelet aggregation by 50%(IC50) was determined.

As shown in FIG. 2, the peptides of the invention were variouslyeffective in inhibiting platelet aggregation (column labelled "Plate.Aggreg. (hep)"). Peptides 754 and 940 were particularly effective ininhibiting platelet aggregation at a low concentrations. These peptidesalso are relatively potent for binding the α_(V) β₃ and, therefore, canbe useful for reducing or preventing restenosis (see Choi et al., supra,1994; Topol et al., supra. 1994).

7. Effect of RGD Peptides on Angiogenesis

This example describes the use of the chick chorioallantoic membrane(CAM) assay to identify peptides of the invention that reduce or inhibitangiogenesis.

The CAM assay is performed as described by Brooks et al., supra, 1994a,1994b). Tumor culture on the chick CAM is accomplished using 10 day oldchick embryos. A small hole is made through the egg shell at the end ofthe egg directly over the sac using a small crafts drill (Dremel; RacineWis.). A second hole is drilled on the broad side of the egg directlyover embryonic blood vessels. Negative pressure is applied to theoriginal hole, resulting in the CAM pulling away from the shell on theopposing side. A 1×1 cm window is cut in the shell and 50 mg fragmentsof human tumor, which are grown on a separate embryo CAM, is placed onthe CAM of the new embryo. The window is covered with tape and theembryo is incubated 24 hr at 37° C.

Following the 24 hr incubation period, the embryo is inoculated with 300μg of a peptide or with saline (control). Incubation then is continuedfor an additional 3 days, after which the tumor is resected and analyzedfor angiogenesis, or for an additional 7 days, after which the tumor isremoved and weighed to determine the change from the original weight.Tumors can be analyzed histologically using standard fixation andparaffin embedding procedures.

EXAMPLE III RGD Peptide Activity In Vivo

This example provides methods for determining the activity of an RGDpeptide of the invention to reduce or inhibit bone resorption orrestenosis in vivo.

A. Effect of RGD Peptides on Parathyroid Hormone-induced Hypercalcemiain Mice

This example demonstrates that the RGD peptides of the invention caninhibit parathyroid hormone-induced hypercalcemia, which is a measure ofosteoclast activation in a subject. Parathyroid hormone (PTH) activatesosteoclasts, which are involved in bone resorption. Associated with theincreased osteoclast activity is an increase in serum calcium. ThePTH-induced hypercalcemia assay measures the level of serum calcium,which is an indication of bone resorption in a subject (Fischer et al.,Endocrinology 132:1411-1413 (1993), which is incorporated herein byreference).

Swiss-Webster mice, which had been parathyroidectomized two weeks priorto beginning the assay, were obtained from Taconic Lab Animals andServices (Germantown N.Y.). Femoral vein catheters (PE-50 fused withPE10 polyethylene tubing; Becton Dickinson; Parsippany, N.J.) wereinstalled in the mice, flushed and locked with heparinized saline(1:10), sutured to adjacent muscle and internalized within the inguinalspace of the incision before closing the incision with wound clips.Baseline serum ionized calcium levels were less than 1.29 mM for miceselected for this study. Serum calcium measurements were performed byatomic absorption spectrophotometry using a Perkin-Elmer model 2380 AAspectrophotometer (Perkin-Elmer; Norwalk Conn.).

The experiment was initiated by feeding the mice a calcium-deficientdiet overnight, then injecting the mice the following morning with abolus of 0.2 mg PTH/kg (hPTH 1-34; Bachem; Torrance Calif.),intravenously. An ionized serum calcium level was obtained 30 min priorto PTH infusion. Various doses of a peptide or vehicle were infused intoan animal for 4 hr, then ionized serum calcium again was measured.Lyophilized peptide was redissolved in PBS or in water, depending on thesolubility at a particular concentration. Serum calcium measurementswere expressed as the difference between the value 30 min before the PTHpulse and 4 hr after peptide infusion was started.

One control peptide used for this experiment was echistatin (Bachem;Torrance Calif.), which was administered at doses ranging from 0-30mg/kg/hr. Another control peptide was TL200, an RGD-containing peptidethat is selective for the GPIIbIIIa integrin receptor present onplatelets. The data presented in FIG. 9 demonstrate a dose responsiveeffect of the peptides. Peptides TL20 and TL191 decreased thePTH-induced rise in serum ionized calcium in a dose responsive manner.Peptide TL386 was analyzed at a single dose, which resulted in arelatively low level of serum calcium measured in animals within thisgroup. The control peptide, TL200, did not show a dose responsiveeffect, confirming the selective nature of the TL20, TL191 and TL 386peptides.

The ability of the peptides of the invention to reduce or inhibit boneresorption also is evaluated by determining the inhibition of bone lossin an ovariectomized rat, which is a model for estrogendeficiency-induced bone loss as occurs in post-menopausal women. Theassay is performed as described by Kalu, Bone Miner. 15:175-192 (1991),which is incorporated herein by reference. In addition, useful animalmodel systems for evaluating the effectiveness of a peptide to reduce orinhibit bone resorption are described by Rogers et al., Bone 14:369-377(1993), which is incorporated herein by reference.

In experiments using these model systems, a peptide is administered toan ovariectomized animal and bone loss is measured over a period of timesuch as over a five week period. A composition comprising the peptidecan be administered in any of various ways such as intravenous,intraperitoneal, intramuscular or subcutaneous injection. In particular,a composition can be administered subcutaneously in the form ofpeptide-containing microcapsules.

The skilled artisan will understand that an effective peptide can beidentified by administering various amounts of a peptide to groups ofanimals in order to obtain statistically significant results. Forexample, a range of peptide doses of about 0.1, 1, 10 or 100 mg/kg/daycan provide an indication of an effective amount of a peptide, which canreduce or inhibit bone resorption. One or more groups of animals canserve as appropriate controls.

B. Effect of RGD Peptides on Restenosis in Guinea Pigs

This example provides a method for identifying RGD peptides of theinvention that can reduce or inhibit restenosis. Various animal modelsof restenosis are well known (see, for example, Choi et al., supra,1994; see, also, Jackson, Trends Cardiovasc. Med. 4:122-130 (1994)). Aguinea pig model of restenosis as described herein also is useful foridentify effective peptides.

Guinea pigs (300-400. g; 2-3 months old) are anesthetized and the leftcommon carotid artery (LCCA) and right internal jugular vein are exposedthrough a midline incision in the neck. The internal vein is isolatedand ligated. A silastic tube (PE-160) attached to an "ALZET" osmoticminipump (2 ml; Alza; Palo Alto Calif.) is introduced into the open endof the same vessel and the pump is placed in a subcutaneous tunnel onthe dorsal surface of the animal. A 2F Fogarty balloon catheter isintroduced into the left external carotid artery (LECA), inflated andwithdrawn three times to denude the endothelium in the LCCA. Thecatheter then is removed, the LECA is ligated and the surgical wound isclamped.

Saline (control) or various concentrations of peptide are delivered viathe minipump at a rate of about 10 μl/hr for 7 days. Three weeks afterballoon injury, guinea pigs are anesthetized and an abdominal incisionis made to expose the abdominal aorta. An 18 gauge IV catheter is usedto flush (50 ml Ringer's lactate) and fix (200 ml 4% formaldehyde) invivo during a lethal injection of pentothal via the tail vein.Histological sections are prepared by cutting the LCCA in serial 2 mmsections and staining with hematoxylin-eosin and with Lawson'selastic-van Gieson stain on separate slides. Morphometric analyses areperformed using a computerized digital microscopy algorithm to measurethe cross-sectional area of the lumen, intima, media and adventitia forthe extent of neointimal thickening.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 138                                                (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ArgGlyAspAspVal                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    N- terminal."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ArgGlyAspAspValGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ArgGlyAspAspValGlu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GlyArgGlyAspSerPro                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GlyArgGlyAspSerProLys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ValThrArgGlyAspValPheThrLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GlyArgGlyAspAspValAsp                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    N- terminal."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CysLysGlyAspAspValCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlyArgGlyAspSerProAspGly                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GlyArgGlyAspSerProAspPhe                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      PheArgGlyAspSerProGluGly                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GlyArgGlyAspThrPheGlu                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ArgGlyAspSerProGlu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GlyArgAlaArgGlyAspAsnProGlu                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ArgGlyAspThrProGlu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GlyArgGlyAspGluProAspGly                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    N- terminal."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CysArgGlyAspAspValCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    N- terminal."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CysIleAlaArgGlyAspAspValCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    N- terminal."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CysGluProArgGlyAspAspValCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    N- terminal."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CysGluAlaArgGlyAspAspValCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    N- terminal."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CysAlaAlaArgGlyAspThrProCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    N- terminal."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      PheCysArgGlyAspThrPheCys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    N- terminal."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CysIlePheArgGlyAspThrPheCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    N- terminal."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      ArgGlyAspAspValGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      ArgGlyAspAsnIleGlu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    N- terminal."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      AlaCysArgGlyAspAspValCysAla                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    N- terminal."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      AlaCysArgGlyAspGluValCysAla                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      ArgGlyAspSer                                                                  (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      GlyArgGlyGluSerPro                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa = Pen"                                     (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      GlyXaaAlaAlaArgGlyAspGluProCys                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "Xaa is Tic"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      ArgGlyAspAsnXaa                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa = R1R2, (wherein R1 is                     an H or alkyl group and R2 is an H, alkyl, CH3CO,                             alkyl-CO or phenyl-CO group) or 0 to 10 amino acids,                          which can be protected by acetylation at the N-terminus."                     (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa = 0 or 1 amino acid"                       (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "Xaa = 0, 1 or 2 amino                          acids"                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "Xaa = a positively charged                     amino acid"                                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Xaa = an amino acid which                      can provide a hydrogen bond interaction with an                               integrin receptor"                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa = an amino acid that                       has the characteristics of hydrophobicity or                                  conformational constraint"                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Xaa = a residue capable of                     forming a bond with a bridging amino acid of Xaa at                           position 2, or with Xaa at position 3 when Xaa at                             position 2 is 0, or with Xaa at position 4 when Xaa                           at position 2 and Xaa at position 3 are 0, to                                 conformationally restrain the peptide."                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 10                                                              NR3R4, (wherein R3ION: /note= "Xaa =                                          is an H or alkyl group and R4 is an H or alkyl                                group) or - OR5 (wherein R5 is an H or alkyl group)                           or 0 to 10 amino acids, which can be protected as an                          amide at the C-terminus, and wherein when Xaa at                              position 7 is serine and Xaa at position 8 is proline,                        Xaa at position 3 is 0 or 2 amino acids."                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      XaaXaaXaaXaaGlyAspXaaXaaXaaXaa                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa = R1R2, (wherein R1 is                     an H or alkyl group and R2 is an H, alkyl, CH3CO,                             alkyl-CO or phenyl-CO group) or 0 to 10 amino acids,                          which can be protected by acetylation at the N-terminus."                     (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa = 0 or 1 amino acid"                       (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "Xaa = 0, 1 or 2 amino                          acids"                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "Xaa = a positively charged                     amino acid"                                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Xaa = an amino acid that                       provides an ionic or similar interaction with an                              integrin receptor, or is Msa, Psa or Tfsa."                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa = an amino acid having                     an aliphatic side chain or is an aliphatic                                    non-natural amino acid that is hydrophobic, or is Thr."                       (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Xaa = a residue capable of                     forming a bond with a bridging amino acid of Xaa at                           position 2, or with Xaa at position 3 when Xaa at                             position 2 is 0, or with Xaa at position 4 when Xaa                           at position 2 and Xaa at position 3 are 0, to                                 conformationally restrain the peptide."                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 10                                                              NR3R4, (wherein R3ION: /note= "Xaa =                                          is an H or alkyl group and R4 is an H or alkyl                                group) or - OR5 (wherein R5 is an H or alkyl group)                           or 0 to 10 amino acids, which can be protected as an                          amide at the C-terminus."                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      XaaXaaXaaXaaGlyAspXaaXaaXaaXaa                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pmc"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GlyXaaArgGlyAspCysAla                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      GlyXaaGlyArgGlyAspAsnTyrCysAla                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      GlyXaaGlyGluArgGlyAspAsnTyrCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      GlyXaaAlaAlaArgGlyAspValProCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      GlyXaaGluLeuArgGlyAspGlyTrpCys                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      GlyXaaGlyPheArgGlyAspGluProCys                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      LysXaaGlyPheArgGlyAspGluProCysArg                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      LysXaaGlyPheArgGlyAspAspProCysArg                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Xaa is Orn"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      XaaAlaAlaArgGlyAspXaaProCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "Xaa is (YOMe)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      XaaArgGlyAspXaaXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "Xaa is (Cha)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      XaaArgGlyAspXaaXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      CysArgGlyAspSerXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      GlyXaaGlyArgGlyAspSerProCysAla                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "Xaa is (YOMe)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      ArgGlyAspXaaArgGlu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      GlyXaaArgAlaArgGlyAspAspLeuCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Xaa is nL"                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      GlyXaaArgAlaArgGlyAspAspXaaCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      GlyXaaAlaAlaArgGlyAspAspIleCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Beta-Ala"                               (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      XaaArgAlaArgGlyAspAsnProAsp                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is Pgl"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      XaaPheAlaArgGlyAspAsnXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "Xaa is (p-Cl-F)"                               (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      XaaIleXaaArgGlyAspThrPheCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "Xaa is Tic"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      ArgGlyAspSerXaaGlu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      GlyXaaGlyHisArgGlyAspSerProCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      GlyXaaArgAlaArgGlyAspAsnProCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pmp"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      XaaArgGlyAspSerProXaaGly                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Xaa is (YOMe)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      CysAsnProArgGlyAspXaaArgCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      GlyXaaAlaAlaArgGlyAspAsnProCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      GlyXaaIlePheArgGlyAspThrPheCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      GlyXaaAlaAlaArgGlyAspSerProCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      GlyXaaIleAlaArgGlyAspAspLeuCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (dhP)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      XaaAlaAlaArgGlyAspAsnXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      CysAlaAlaArgGlyAspAsnXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      GlyXaaArgAlaArgGlyAspAspValCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      XaaPheAlaArgGlyAspAsnProCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      GlyXaaGluAlaArgGlyAspAsnProCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      CysArgGlyAspAsnProXaa                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      XaaAlaAlaArgGlyAspAsnIleCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:70:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                      XaaAlaAlaArgGlyAspAsnXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:71:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                      XaaThrAlaArgGlyAspAsnProCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:72:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                      GlyXaaAlaAlaArgGlyAspAspValCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:73:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                      CysAlaAlaArgGlyAspTyrXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:74:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                      XaaPheAlaArgGlyAspSerProCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:75:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is (YOMe)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                      XaaXaaAlaArgGlyAspAsnProCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:76:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (p-Cl-F)"                               (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                      XaaArgGlyAspThrProAsp                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:77:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                      XaaPheAlaArgGlyAspAsnXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:78:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                      CysArgGlyAspSerProCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:79:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                      GlyXaaAlaAlaArgGlyAspAspThrCys                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:80:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is (YOMe)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                      XaaXaaAlaArgGlyAspAsnXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:81:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Mpa"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                      XaaArgGlyAspAspValCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:82:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                      XaaPheAlaArgGlyAspSerXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:83:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                      CysAlaAlaArgGlyAspSerXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:84:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is beta-Alanine"                           (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                      XaaAlaAlaArgGlyAspAsnXaaAsp                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:85:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                      CysAlaAlaArgGlyAspThrXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:86:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                      GlyCysGluProArgGlyAspAsnXaaCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:87:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Xaa is (Pen)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                      CysArgGlyAspSerXaaXaa                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:88:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                      CysArgGlyAspThrXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:89:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (1-Nal)"                                (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                      XaaAlaAlaArgGlyAspAsnXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:90:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (fp)"                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                      CysAlaAlaArgGlyAspAsnXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:91:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Xaa is (Pen)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                      CysArgGlyAspAsnXaaXaa                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:92:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                      GlyXaaGlyPheArgGlyAspSerProCys                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:93:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                      CysArgGlyAspSerXaaCysAlaLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:94:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                      XaaPheAlaArgGlyAspAspValCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:95:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is (2-Nal)"                                (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                                      XaaXaaAlaArgGlyAspAsnProCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:96:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (7-OMeTic)"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                                      CysAlaAlaArgGlyAspAsnXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:97:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Pen)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                                      AlaCysArgGlyAspSerProXaa                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:98:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                                      CysAlaAlaArgGlyAspAsnXaaCysLys                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:99:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "Xaa is (nMeF)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                                      XaaIleXaaArgGlyAspThrPheCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:100:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 17                                                              (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:                                     ThrAlaProGlyLysHisProAsnArgCysAlaAlaArgGlyAspAsn                              151015                                                                        XaaCys                                                                        (2) INFORMATION FOR SEQ ID NO:101:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is Pen"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (PgL)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:                                     XaaPheAlaArgGlyAspAspXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:102:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:                                     CysPheAlaArgGlyAspThrXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:103:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (t-BuG)"                                (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:                                     XaaArgGlyAspAspXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:104:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (cha)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:                                     XaaArgGlyAspAspXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:105:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:                                     ArgGlyAspAsnXaaGlu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:106:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:                                     CysAlaAlaArgGlyAspAsnXaaCysAlaLys                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:107:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tca)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:                                     CysArgGlyAspThrXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:108:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (Ole)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:                                     CysArgGlyAspThrXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:109:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Pen)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:                                     XaaGluAlaArgGlyAspAspValCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:110:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:                                     GlyAlaAlaArgGlyAspSerXaaGlu                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:111:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Pen)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (homoP)"                                (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:                                     XaaAlaAlaArgGlyAspAsnXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:112:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "Xaa is (Adp)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:                                     ArgGlyAspSerXaa                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:113:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:                                     CysGluProArgGlyAspAsnXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:114:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Pmp)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:                                     XaaAlaAlaArgGlyAspAspAsnXaaCys                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:115:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Adp)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:                                     AlaAlaArgGlyAspAspValXaa                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:116:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Pen)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:                                     XaaGluProArgGlyAspAspValCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:117:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (npG)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:                                     XaaArgGlyAspAspXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:118:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Pen)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:                                     XaaIleGlyArgGlyAspAspXaaCysAla                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:119:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:                                     XaaArgGlyAspAspXaaCysAla                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:120:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "Xaa is (tetA)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:                                     XaaArgGlyAspXaaXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:121:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "Xaa is (HomoR)"                                (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:                                     CysAlaAlaXaaArgGlyAspAsnXaaCys                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:122:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is (nMeR)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:                                     CysXaaGlyAspThrXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:123:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:                                     XaaArgGlyAspAspXaaCysAlaLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:124:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:                                     XaaArgGlyAspAspXaaCysAsp                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:125:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:                                     PheCysArgGlyAspThrXaaCys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:126:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "Xaa is (Msa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:                                     XaaArgGlyAspXaaXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:127:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:                                     GlyArgGlyAspAspXaaGlu                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:128:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:                                     XaaArgGlyAspAspXaaCysPhe                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:129:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Beta-Ala)"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:                                     XaaArgGlyAspAspXaaAsp                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:130:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is (nMeR)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:                                     XaaXaaGlyAspAspXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:131:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "Xaa is (Psa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:                                     XaaArgGlyAspXaaXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:132:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "Xaa is (YOMe)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      C- terminal."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:                                     XaaArgGlyAspXaaXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:133:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:                                     XaaArgGlyAspAspValCysAlaLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:134:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (PgL)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:                                     XaaArgGlyAspAspXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:135:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mamb)"                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:                                     ArgGlyAspAspXaaXaa                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:136:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Mpa)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tlsa)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Xaa is (tBuG)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:                                     XaaArgGlyAspXaaXaaCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:137:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Amino acid is acetylated at                    the N- terminal."                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is (Pen)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "Xaa is (YOMe)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa is (Tic)"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:                                     XaaXaaAlaArgGlyAspThrXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:138:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "Xaa is (YOMe)"                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "Amino acid is amidated at                      the C- terminal."                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:                                     ArgGlyAspXaaArgGlu                                                            15                                                                            __________________________________________________________________________

We claim:
 1. A method of altering bone resorption in a subject in needthereof, comprising administering to the subject a peptide having thestructure:

    X.sub.1 X.sub.2 X.sub.3 X.sub.4 GD X.sub.5 X.sub.6 X.sub.7 X.sub.8

wherein: X₁ is R₁ R₂, wherein R₁ is an H or alkyl group and R₂ is an H,alkyl, CH₃ CO, alkyl-CO or phenyl-CO group; or 0 to 10 amino acids,which can be protected by acetylation at an N-terminus; X₂ is 0 or 1amino acid; X₃ is 0, 1 or 2 amino acids; X₄ is N-Me-Arg; X₅ is an aminoacid that provides an ionic interaction with an integrin receptor or isMsa, Psa or Tfsa; X₆ is an amino acid having an aliphatic side chain oris a non-natural amino acid that is hydrophobic or is Thr; X₇ is aresidue capable of forming a bond with a bridging amino acid of X₂ ; orwith X₃ when X₂ is 0; or with X₄ when X₂ and X₃ are 0, toconformationally restrain the peptide; X₈ is --NR₃ R₄, wherein R₃ is anH or alkyl group and R₄ is an H or alkyl group; or --OR₅, wherein R₅ isan H or alkyl group; or 0 to 10 amino acids, which can be protected asan amide at the C-terminus; and, wherein bone resorption is altered. 2.A method of altering osteoclast binding to a matrix in a subject in needthereof, comprising contacting the osteoclast with an effective amountof a peptide having the structure:

    X.sub.1 X.sub.2 X.sub.3 X.sub.4 GD X.sub.5 X.sub.6 X.sub.7 X.sub.8

wherein: X₁ is R₁ R₂, wherein R₁ is an H or alkyl group and R₂ is an H,alkyl, CH₃ CO, alkyl-CO or phenyl-CO group; or 0 to 10 amino acids,which can be protected by acetylation at an N-terminus; X₂ is 0 or 1amino acid; X₃ is 0, 1 or 2 amino acids; X₄ is N-Me-Arg; X₅ is an aminoacid that provides an ionic interaction with an integrin receptor or isMsa, Psa, or Tfsa; X₆ is an amino acid having an aliphatic side chain oris a non-natural amino acid that is hydrophobic or is Thr; X₇ is aresidue capable of forming a bond with a bridging amino acid of X₂ ; orwith X₃ when X₂ is 0; or with X₄ when X₂ and X₃ are 0, toconformationally restrain the peptide; X₈ is --NR₃ R₄, wherein R₃ is anH or alkyl group and R₄ is an H or alkyl group; or --OR₅, wherein R₅ isan H or alkyl group; or 0 to 10 amino acids, which can be protected asan amide at the C-terminus; and, wherein osteoclast binding to a matrixis altered.